Prokaryotes

J Biochem Mol Biol, 2002 Jul 31, 35(4), 437 - 41
Activity of human dihydrolipoamide dehydrogenase is reduced by mutation at threonine-44 of FAD-binding region to valine; Kim H; Dihydrolipoamide dehydrogenase (E3) is a member of the pyridine nucleotide-disulfide oxidoreductase family . Thr residues are highly conserved . They are at the active site disulfide-bond regions of most E3s and other oxidoreductases . The crystal structure of Azotobacter vinelandii E3 suggests that the hydroxyl group of Thr that are involved in the FAD binding interact with the adenosine phosphate of FAD . However, several prokaryotic E3s have Val instead of Thr . To investigate the meaning and importance of the Thr conservation in many E3s, the corresponding residue, Thr-44, in human E3 was substituted to Val by site-directed mutagenesis . The mutant's E3 activity showed about a 2.2-fold decrease . Its UV-visible and fluorescence spectra indicated that the mutant might have a slightly different microenvironment at the FAD-binding region.

Environ Med, 1999 Dec, 43(2), 79 - 87
The role of heat shock proteins in mammalian differentiation and development; Walsh D et al.; Heat shock proteins (HSPs) have been identified in all cells, prokaryotic and eukaryotic, to protect the cells from harmful insults and stress . Increased HSP synthesis can also result during normal cellular functions and also respond to exposure from environmental stress and infection . Although the molecular mechanisms responsible for HSP cellular protection are still not fully understood, their expression is critical for cellular survival and can be modified by cell signal transducers such as intracellular pH, cyclic AMP, Ca2+ Na+, inositol {correction of inostitol} triphosphate, protein kinase C, and protein phosphates . Most HSPs interact, assemble, fold, unfold, bind, transport, translocate and 'chaperone' other proteins in the cell and alter their function.

J Bacteriol, 2002 Oct, 184(20), 5733 - 45
Correlations between Shine-Dalgarno sequences and gene features such as predicted expression levels and operon structures; Ma J et al.; This work assesses relationships for 30 complete prokaryotic genomes between the presence of the Shine-Dalgarno (SD) sequence and other gene features, including expression levels, type of start codon, and distance between successive genes . A significant positive correlation of the presence of an SD sequence and the predicted expression level of a gene based on codon usage biases was ascertained, such that predicted highly expressed genes are more likely to possess a strong SD sequence than average genes . Genes with AUG start codons are more likely than genes with other start codons, GUG or UUG, to possess an SD sequence . Genes in close proximity to upstream genes on the same coding strand in most genomes are significantly higher in SD presence . In light of these results, we discuss the role of the SD sequence in translation initiation and its relationship with predicted gene expression levels and with operon structure in both bacterial and archaeal genomes.

J Bacteriol, 2002 Oct, 184(20), 5641 - 53
Adaptive, or stationary-phase, mutagenesis, a component of bacterial differentiation in Bacillus subtilis; Sung HM et al.; Adaptive (stationary-phase) mutagenesis occurs in the gram-positive bacterium Bacillus subtilis . Furthermore, taking advantage of B . subtilis as a paradigm for the study of prokaryotic differentiation and development, we have shown that this type of mutagenesis is subject to regulation involving at least two of the genes that are involved in the regulation of post-exponential phase prokaryotic differentiation, i.e., comA and comK . On the other hand, a functional RecA protein was not required for this type of mutagenesis . The results seem to suggest that a small subpopulation(s) of the culture is involved in adaptive mutagenesis and that this subpopulation(s) is hypermutable . The existence of such a hypermutable subpopulation(s) raises important considerations with respect to evolution, the development of specific mutations, the nature of bacterial populations, and the level of communication among bacteria in an ecological niche.

J Bacteriol, 2002 Oct, 184(20), 5554 - 62
Distinct clpP genes control specific adaptive responses in Bacillus thuringiensis; Fedhila S et al.; ClpP and ClpC are subunits of the Clp ATP-dependent protease, which is ubiquitous among prokaryotic and eukaryotic organisms . The role of these proteins in stress tolerance, stationary-phase adaptive responses, and virulence in many bacterial species has been demonstrated . Based on the amino acid sequences of the Bacillus subtilis clpC and clpP genes, we identified one clpC gene and two clpP genes (designated clpP1 and clpP2) in Bacillus thuringiensis . Predicted proteins ClpP1 and ClpP2 have approximately 88 and 67% amino acid sequence identity with ClpP of B . subtilis, respectively . Inactivation of clpC in B . thuringiensis impaired sporulation efficiency . The clpP1 and clpP2 mutants were both slightly susceptible to salt stress, whereas disruption of clpP2 negatively affected sporulation and abolished motility . Virulence of the clp mutants was assessed by injecting bacteria into the hemocoel of Bombyx mori larvae . The clpP1 mutant displayed attenuated virulence, which appeared to be related to its inability to grow at low temperature (25 degrees C), suggesting an essential role for ClpP1 in tolerance of low temperature . Microscopic examination of clpP1 mutant cells grown at 25 degrees C showed altered bacterial division, with cells remaining attached after septum formation . Analysis of lacZ transcriptional fusions showed that clpP1 was expressed at 25 and 37 degrees C during the entire growth cycle . In contrast, clpP2 was expressed at 37 degrees C but not at 25 degrees C, suggesting that ClpP2 cannot compensate for the absence of ClpP1 in the clpP1 mutant cells at low temperature . Our study demonstrates that ClpP1 and ClpP2 control distinct cellular regulatory pathways in B . thuringiensis.

Biochemistry, 2002 Oct 1, 41(39), 11750 - 60
Regulation of actin dynamics by tyrosine phosphorylation: identification of tyrosine phosphorylation sites within the actin-severing domain of villin; Zhai L et al.; We have previously shown that villin, an epithelial cell actin-binding protein, is tyrosine phosphorylated both in vitro and in vivo and that villin's actin-modifying functions are regulated by phosphorylation . Here as a first step toward understanding the role of villin tyrosine phosphorylation, we sought to identify the major phosphorylation site(s) in human villin and study its role in actin filament assembly . We generated a series of carboxyl-terminal truncation mutants of villin and cloned them in the prokaryotic expression vector pGEX-2T . Full-length villin and the truncation mutants were expressed in TKX1 cells, which carry an inducible tyrosine kinase gene . Using this approach, we identified a region in the amino-terminal actin-severing domain of villin as the site of phosphorylation (amino acids 1-261) . Five phosphorylation sites were identified by direct mutation of candidate tyrosines (Y) to phenylalanine (F), namely, Y46, -60, -64, -81, and -256 . Changing all of these sites to phenylalanine resulted in a villin mutant that neither was phosphorylated in TKX1 cells nor was a substrate for c-src kinase in an in vitro kinase assay . Using a pyrene actin-based fluorescence assay, we mapped the various phosphorylated tyrosine residues with the actin-nucleating and -depolymerizing functions of villin . Phosphorylation of any one of the identified sites inhibited the actin-nucleating function of villin, whereas phosphorylation at Y46 and/or Y60 increased the actin-severing activity of villin . Since there is significant homology between the amino-terminal end of villin and other actin-severing proteins, the results provide a structural basis for the actin-severing mechanism and help understand the relationship of phosphorylation with this function.

Biochemistry, 2002 Oct 1, 41(39), 11703 - 10
Structural and energetic analysis of metal ions essential to SRP signal recognition domain assembly; Batey RT et al.; The signal recognition particle (SRP) targets proteins to the endoplasmic reticulum in eukaryotes or to the inner membrane in prokaryotes by binding to hydrophobic signal sequences . Signal peptide recognition occurs within the highly conserved RNA-protein core of the SRP, underscoring the importance of this complex in SRP function . Structural analysis of the RNA and protein components of the prokaryotic SRP in the free and bound states revealed that the RNA undergoes a significant conformational change upon protein binding involving the uptake of several monovalent and divalent cations . To investigate the role of these metal ions in formation of the functional SRP complex, we used binding affinity assays and X-ray crystallography to analyze the specificity and energetic contributions of mono- and divalent metal ions bound in the RNA . Our results demonstrate that several metal ion binding sites important for RNA conformation can accommodate chemically distinct ions, often without affecting the structure of the complex . Thus, while these metal ions are highly ordered and essential for the formation and stability of the SRP complex, they behave like nonspecific metal ions.

Poult Sci, 2002 Sep, 81(9), 1317 - 21
Stimulatory effect of CpG sequences on humoral response in chickens; Vleugels B et al.; Oligonucleotides containing the dinucleotide CpG have an immunostimulatory effect in mammals . The CpG motif is interpreted as a signal of prokaryote invasion by the innate immune system and consequently activates defense mechanisms . The goal of this study was to investigate whether the immunostimulatory actions of CpG oligonucleotides take place in birds as well . To this end, birds were immunized with BSA and the serum antibody response was followed . A significantly higher BSA-specific response was observed in the CpG-treated group . Moreover, immunostimulatory DNA resulted in more persistent responses to immunization . After only one immunization, titers as high as in booster responses were observed in the CpG-treated birds . The effects were shown to depend on the presence of an unmethylated CpG core sequence in the DNA, because the reversal of CpG to GpC even caused a decrease in antibody response . These findings demonstrate that CpG oligonucleotides could be a valuable adjuvant for poultry vaccines.

Genetics, 2002 Sep, 162(1), 217 - 27
Identification and analysis of a hyperactive mutant form of Drosophila P-element transposase; Beall EL et al.; Transposition in many organisms is regulated to control the frequency of DNA damage caused by the DNA breakage and joining reactions . However, genetic studies in prokaryotic systems have led to the isolation of mutant transposase proteins with higher or novel activities compared to those of the wild-type protein . In the course of our study of the effects of mutating potential ATM-family DNA damage checkpoint protein kinase sites in the Drosophila P-element transposase protein, we found one mutation, S129A, that resulted in an elevated level of transposase activity using in vivo recombination assays, including P-element-mediated germline transformation . In vitro assays for P-element transposase activity indicate that the S129A mutant exhibits elevated donor DNA cleavage activity when compared to the wild-type protein, whereas the strand-transfer activity is similar to that of wild type . This difference may reflect the nature of the in vitro assays and that normally in vivo the two reactions may proceed in concert . The P-element transposase protein contains 10 potential consensus phosphorylation sites for the ATM family of PI(3)-related protein kinases . Of these 10 sites, 8 affect transposase activity either positively or negatively when substituted individually with alanine and tested in vivo . A mutant transposase protein that contains all eight N-terminal serine and threonine residues substituted with alanine is inactive and can be restored to full activity by substitution of wild-type amino acids back at only 3 of the 8 positions . These data suggest that the activity of P-element transposase may be regulated by phosphorylation and demonstrate that one mutation, S129A, results in hyperactive transposition.

Biochem J, 2002 Oct 1, 367(Pt 1), 219 - 27
Cloning and characterization of anti-cathepsin L single chain variable fragment whose expression inhibits procathepsin L secretion in human melanoma cells; Guillaume-Rousselet N et al.; We previously demonstrated that increase of procathepsin L secretion by human melanoma cells strongly increased their tumourigenicity and switched their phenotype from low to highly metastatic . Thus, we herein analysed whether it was possible to inhibit procathepsin L secretion using anti-cathepsin L ScFv . For this purpose, we produced different forms of fusion cathepsin L in prokaryotic or eukaryotic expression systems . An anti-cathepsin L monoclonal antibody (mAb), named 3D8, was isolated from mice immunized with purified procathepsin L-His . This 3D8 mAb interacted with an epitope localized on the 156-197 amino acid sequence of cathepsin L and recognized recombinant or native forms of cathepsin L synthesized by human melanoma cells . An active anti-cathepsin L ScFv was generated and characterized from 3D8 mAb heavy and light variable chains . Then, human melanoma cells were transiently co-transfected with 3D8 ScFv and cathepsin L cDNAs . Data demonstrated that increase of 3D8 ScFv expression in human melanoma cells totally inhibited procathepsin L secretion and induced accumulation of intracellular procathepsin L . Our results constitute the first demonstration that anti-cathepsin L ScFv could be used in human melanoma cells to inhibit procathepsin L secretion . This ScFv represents a new molecular tool to explore cell therapy of human melanomas.

Mol Biol Rep, 2002, 29(1-2), 227 - 32
Virtual mitochondria: metabolic modelling and control; Aimar-Beurton M et al.; Inside the eukaryotic cell, mitochondria are internal organelles of prokaryotic origin, responsible for energy supply in the cell . The control of the mitochondrial ATP production is a complex problem with different patterns according to different tissues and organs . Our aim is to continue to develop the modelling of oxidative phosphorylation in different tissues, to model other parts of mitochondrial metabolism and to include this virtual mitochondria in a virtual cell . In constructing the complete metabolic map of mitochondria, we will take advantage of the sequenced genomes of eukaryotic organism (10-15% of the yeast genome concerns mitochondria).

Biotechniques, 2002 Sep, 33(3), 672, 674, 676 - 8
Development of multiple cloning site cis-vectors for recombinant adeno-associated virus production; Yue Y et al.; Recombinant adeno-associated virus (rAAV) has become a very popular gene therapy vector in the past several years . A cis-plasmid is used to generate the rAAV stocks . In this plasmid, the entire expression cassette is incorporated between two AAV inverted terminal repeats . The construction of cis-plasmid has been problematic because of the high-frequency recombination of the viral inverted terminal repeats . Here we describe the design and construction of several multiple cloning site cis-plasmids that are driven by five different promoters, including the ubiquitous cytomegalovirus enhancer/chicken beta-actin (CAG), cytomegalovirus (CMV), rous sarcoma virus (RSV), simian virus 40 (SV40), and a muscle-specific promoter (CK6) . The application of these multiple cloning site cis-plasmids improves the cloning efficiency . As an example of the utilization of these multiple cloning site vectors, the prokaryotic beta-galactosidase cDNA was cloned in the multiple cloning site cis-plasmids . High-level rAAV-mediated beta-galactosidase expression was achieved in HeLa cells from CAG, CMV, RSV and SV40 promoters, respectively, but notfrom the CK6 promoter . In vivo application in the adult mdx mouse (mouse model for Duchenne muscular dystrophy) muscle revealed efficient transgene expression from CMV and CK6 promoters, followed by CAG and RSV promoters . The SV40 promoter was the least efficient.

Biotechniques, 2002 Sep, 33(3), 548 - 50, 552, 554-6
GWFASTA: server for FASTA search in eukaryotic and microbial genomes; Issac B et al.; Similarity searches are a powerful method for solving important biological problems such as database scanning, evolutionary studies, gene prediction, and protein structure prediction . FASTA is a widely used sequence comparison tool for rapid database scanning . Here we describe the GWFASTA server that was developed to assist the FASTA user in similarity searches against partially and/or completely sequenced genomes . GWFASTA consists of more than 60 microbial genomes, eight eukaryote genomes, and proteomes of annotatedgenomes . Infact, it provides the maximum number of databases for similarity searching from a single platform . GWFASTA allows the submission of more than one sequence as a single query for a FASTA search . It also provides integrated post-processing of FASTA output, including compositional analysis of proteins, multiple sequences alignment, and phylogenetic analysis . Furthermore, it summarizes the search results organism-wise for prokaryotes and chromosome-wise for eukaryotes . Thus, the integration of different tools for sequence analyses makes GWFASTA a powerful toolfor biologists.

Southeast Asian J Trop Med Public Health, 2002 Jun, 33(2), 288 - 96
Recombinant envelope protein of HIV-1 subtype E as antigen in HIV-1 antibody detection enzyme immunoassay; Sutthent R et al.; In order to develop a reliable and inexpensive serodiagnostic method to be used for anti-HIV antibody detection in Thailand, recombinant envelope (TM or gp41 subunit) protein of HIV-1 subtype E was produced from prokaryotic cell (Escherichia coli) as the source of antigen in enzyme immunoassay (TE diagnostic EIA kit) . HIV-1 gp41 subunit of subtype E was successfully expressed in E . coli in the form of polyhistidine-tagged proteins, comprising of rgp41A (601 bases N-terminal half of TM or 25kDa) and rgp41B (560 bases C-terminal half of TM or 24 kDa) by using an expression vector, pBAD/His C . The amount of protein, dilution of sera, and anti-human IgG labeled HRP used in the EIA test optimized by a checker board titration of the protein and seropositive or seronegative sera, were 5.0 microg/ml, 1:300, and 1:4,000, respectively . The blinded test evaluation of TE-diagnostic EIA in 500 seropositive and 500 seronegative sera which have been simultaneously tested by two available commercial kits and compared with our TE diagnostic EIA, gave 99.6% sensitivity and specificity . The other known genetic subtypes sera such as subtype A (n=5), B (n=9), C (n=4) and D (n=5) were also positive with this EIA . The estimated manufacturer cost per test of rgp41 based anti-HIV antibody detection EIA or TE-diagnostic EIA was about 15 baht . This recombinant envelope (gp41 or TM) protein from HIV-1, which can be produced in large quantities without any hazards from growing the virus and has lower cost to produce anti-HIV antibody serological diagnostic kit, should be considered as an HIV screening test in Thailand.

FASEB J, 2002 Oct, 16(12), 1639 - 41 Epub 2002 Aug 07.
The alpha-like RNA polymerase II core subunit 3 (RPB3) is involved in tissue-specific transcription and muscle differentiation via interaction with the myogenic factor myogenin; Corbi N et al.; RNA polymerase II core subunit 3 (RPB3) is an a-like core subunit of RNA polymerase II (pol II) . It is selectively down-regulated upon treatment with doxorubicin (dox) . Due to the failure of skeletal muscle cells to differentiate when exposed to dox, we hypothesized that RPB3 is involved in muscle differentiation . To this end, we have isolated human muscle RPB3-interacting proteins by using yeast two-hybrid screening . It is of interest that an interaction between RPB3 and the myogenic transcription factor myogenin was identified . This interaction involves a specific region of RPB3 protein that is not homologous to the prokaryotic a subunit . Although RPB3 contacts the basic helix-loop-helix (HLH) region of myogenin, it does not bind other HLH myogenic factors such as MyoD, Myf5, and MRF4 . Coimmunoprecipitation experiments indicate that myogenin contacts the pol II complex and that the RPB3 subunit is responsible for this interaction . We show that RPB3 expression is regulated during muscle differentiation . Exogenous expression of RPB3 slightly promotes myogenin transactivation activity and muscle differentiation, whereas the region of RPB3 that contacts myogenin, when used as a dominant negative molecule (Sud), counteracts these effects . These results indicate for the first time that the RPB3 pol II subunit is involved in the regulation of tissue-specific transcription.

J Theor Biol, 2002 Aug 21, 217(4), 479 - 92
A study of the middle-scale nucleotide clustering in DNA sequences of various origin and functionality, by means of a method based on a modified standard deviation; Nikolaou C et al.; The deviation from randomness in the distribution of nucleotides in genomic sequences is quantified and studied, using a modified standard deviation (MSD) . This method implies a "per block" computation of the standard deviation of the nucleotide frequencies of occurrence, using local means (means taken in a neighborhood of each block) . This quantity may serve as a scale-dependent measure of the nucleotide clustering . In the present work, the meso-scale of tenths of nucleotides is principally explored, by means of suitably adjusted filter parameters . This length scale is of an order of magnitude not directly affected by the grammar and syntax rules of the protein-coding procedure, remaining shorter than the scale of appearance of large-scale characteristics of the genome . MSD has been found to distinguish systematically between the sequences of different origin and functionality . The most near-random are found to be coding sequences of prokaryotes, while in intronic and intergenic regions of eukaryotic genomes, extended clustering of similar nucleotides is observed . The distributions of MSD values of large collections of sequences are found to be in most cases characteristic of their biological role and origin . Protein- and non-coding, prokaryotic and eukaryotic DNA as well as promoter, rRNA, viral and organelle sequences have been examined . The presented results corroborate a recently proposed model for genome evolution . The method is also applied for an assessment of the annotation of ORFs taken from the complete genome of Saccharomyces cerevisiae.

Curr Microbiol, 2002 Nov, 45(5), 355 - 61
A conserved Ala320 in the FtsZ of Porphyromonas gingivalis is important for cell division; Yu W et al.; We have previously cloned the gene encoding the cell division protein FtsZ, designated PgFtsZ, from Porphyromonas gingivalis, an oral anaerobic bacterium implicated in advanced periodontal disease . In the present study, we have shown that overexpression of ZDeltaC02, a mutant form of PgFtsZ in which 128 amino acid residues have been removed from the C-terminus, caused an inhibition of cell division in E . coli cells . However, overexpression of ZDeltaC03, missing 177 residues from the C-terminus, did not inhibit cell division, suggesting that the 49 residues between 281 and 329 are required for cell division . Sequence comparison of the known prokaryotic FtsZs revealed that this region contained a highly conserved domain, designated A-domain, in which Ala320 of PgFtsZ was conserved throughout a broad variety of species . Therefore, we analyzed the role of Ala320 by site-directed mutagenesis . We found that overexpression of ZA320H and ZA320R resulted in the normal phenotype, unlike the wild type . Immunoblot analysis showed that these mutant proteins were expressed at similar levels . These results suggest that Ala320 is highly conserved and is crucial for cell division.

Plant Physiol, 1994 Sep, 106(1), 143 - 150
A Mutant of Arabidopsis Deficient in the Elongation of Palmitic Acid; Wu J et al.; The overall fatty acid composition of leaf lipids in a mutant of Arabidopsis thaliana was characterized by an increased level of 16:0 and a concomitant decrease of 18-carbon fatty acids as a consequence of a single recessive nuclear mutation at the fab1 locus . Quantitative analysis of the fatty acid composition of individual lipids established that lipids synthesized by both the prokaryotic and eukaryotic pathways were affected by the mutation . Direct enzyme assays demonstrated that the mutant plants were deficient in the activity of 3-ketoacyl-acyl carrier protein synthase II; therefore, it is inferred that fab1 may encode this enzyme . Labeling experiments with {14C}acetate and lipase positional analysis indicated that the mutation results in a small shift in the partitioning of lipid synthesis between the prokaryotic and eukaryotic pathways . Synthesis of chloroplast lipids by the prokaryotic pathway was increased with a corresponding reduction in the eukaryotic pathway.

Plant Physiol, 1993 Nov, 103(3), 733 - 739
Purification and Properties of a Monofunctional Imidazoleglycerol-Phosphate Dehydratase from Wheat; Mano J et al.; Imidazoleglycerol-phosphate dehydratase (EC 4.2.1.19) activity was detected in extracts of several monocotyledonous and dicotyledonous plants using a newly developed assay method . The enzyme was purified 114,000-fold (to apparent homogeneity) from wheat germ by five chromatographic steps . Its native relative molecular weight (Mr) was determined to be 600,000 to 670,000, and it consists of identical subunits of Mr 25,500 . In wheat germ, the dehydratase, unlike those of prokaryotic origin, is not associated with histidinol phosphatase activity . The reaction product was identified as imidazoleacetol phosphate (IAP) by comparing it with synthetic IAP as an authentic reference . The Km value for imidazoleglycerol phosphate was 0.36 mM at the optimal pH of 6.6 . The enzyme required a reducing agent, such as 2-mercaptoethanol or dithiothreitol, and Mn2+ for maximal activity . 3-Amino-1,2,4-triazole competitively inhibited the activity with a Ki value of 46 {mu}M . The purification of imidazoleglycerol-phosphate dehydratase from wheat germ and histidinol dehydrogenase from cabbage (A . Nagai, A . Scheidegger {1991} Arch Biochem Biophys 284: 127-132) suggests that at least the second half of the histidine biosynthesis in plants is identical to that in microorganisms.

Acta Pol Pharm, 2002 May-Jun, 59(3), 199 - 207
Synthesis of 6-substituted 6H-indolo{2,3-b}quinolines as novel cytotoxic agents and topoisomerase II inhibitors; Kaczmarek L et al.; A systematic investigation into the impact of the substituents introduced into the indolo{2,3-b}quinoline system is described . The findings clearly demonstrate that the compounds bearing a methyl group or a longer aliphatic chain at the N-6 position are inactive against prokaryotic and eukaryotic cells . The introduction of alkyl-amino-alkyl substituent at the N-6 position of indolo{2,3-b}quinoline accounts for the appearance of the antimicrobial and.cytotoxic properties . The cytotoxicity against oral epidermoid carcinoma KB (ID50) is in the range from 2.0 to 9.0 microM, and the antimicrobial activity (MIC) falls between 0.03 and 0.50 mM . The structural relation within 6H-indolo{2,3-b}quinolines, concerning their antimicrobial and cytotoxic activity, corresponds well with their ability to bind DNA and to inhibit topoisomerase II activity.

Infect Immun, 2002 Oct, 70(10), 5827 - 34
Isolation of Chlamydia pneumoniae clonal variants by a focus-forming assay; Gieffers J et al.; Chlamydia pneumoniae is an obligate intracellular prokaryotic human pathogen that causes community-acquired respiratory infection and has been associated with atherosclerosis and cardiovascular disease . Unexpected results from genomic sequencing indicate that significant intrastrain polymorphism exists for some C . pneumoniae isolates . These polymorphisms could reflect genotypes with differing disease-causing characteristics . A definitive means to test this hypothesis is to obtain genetically homogeneous clonal populations of the pathogen and test them in models of infection and disease . To date, methods for cloning C . pneumoniae have not been reported . In this study, we describe the isolation of clonal variants with genetic differences in the tyrP locus from a polymorphic respiratory isolate, using a novel focus-forming assay . These results now allow investigations on the biology and pathogenesis of C . pneumoniae clonal genovars that could lead to new insights into the pathogenesis of this important human pathogen.

J Biol Chem, 2002 Dec 6, 277(49), 47325 - 30 Epub 2002 Sep 11.
X-ray structure of two crystalline forms of a streptomycete ribonuclease with cytotoxic activity; Sevcik J et al.; Ribonuclease (RNase) Sa3 is secreted by the Gram-positive bacterium Streptomyces aureofaciens . The enzyme catalyzes the cleavage of RNA on the 3' side of guanosine residues . Here, x-ray diffraction analysis was used to determine the three-dimensional structure of two distinct crystalline forms of RNase Sa3 to a resolution of 2.0 and 1.7 A . These two structures are similar to each other as well as to that of a homolog, RNase Sa . All of the key active-site residues of RNase Sa (Asn(42), Glu(44), Glu(57), Arg(72), and His(88)) are located in the putative active site of RNase Sa3 . Also herein, RNase Sa3 is shown to be toxic to human erythroleukemia cells in culture . Like onconase, which is an amphibian ribonuclease in Phase III clinical trials as a cancer chemotherapeutic, RNase Sa3 is not inhibited by the cytosolic ribonuclease inhibitor protein . Thus, a prokaryotic ribonuclease can be toxic to mammalian cells.

Curr Infect Dis Rep, 2002 Oct, 4(5), 387 - 399
The Pathophysiology and Treatment of Candida Sepsis; Spellberg B et al.; Sepsis can occur during disseminated candidiasis, but its pathogenesis differs from that caused by typical prokaryotic pathogens . Complex interactions between defects in host defense and "relative" virulence factors expressed by Candida lead to dissemination of the saprophyte to parenchymal organs, and subsequently to onset of multiorgan failure . This review focuses first on the pathophysiology of Candida sepsis, detailing current understanding of host-pathogen interactions . We then consider the choice of antifungal and supportive treatments.

Plant Physiol, 2002 Sep, 130(1), 477 - 86
Cloning and functional characterization of a beta-pinene synthase from Artemisia annua that shows a circadian pattern of expression; Lu S et al.; Artemisia annua plants produce a broad range of volatile compounds, including monoterpenes, which contribute to the characteristic fragrance of this medicinal species . A cDNA clone, QH6, contained an open reading frame encoding a 582-amino acid protein that showed high sequence identity to plant monoterpene synthases . The prokaryotically expressed QH6 fusion protein converted geranyl diphosphate to (-)-beta-pinene and (-)-alpha-pinene in a 94:6 ratio . QH6 was predominantly expressed in juvenile leaves 2 weeks postsprouting . QH6 transcript levels were transiently reduced following mechanical wounding or fungal elicitor treatment, suggesting that this gene is not directly involved in defense reaction induced by either of these treatments . Under a photoperiod of 12 h/12 h (light/dark), the abundance of QH6 transcripts fluctuated in a diurnal pattern that ebbed around 3 h before daybreak (9th h in the dark phase) and peaked after 9 h in light (9th h in the light phase) . The contents of (-)-beta-pinene in juvenile leaves and in emitted volatiles also varied in a diurnal rhythm, correlating strongly with mRNA accumulation . When A . annua was entrained by constant light or constant dark conditions, QH6 transcript accumulation continued to fluctuate with circadian rhythms . Under constant light, advanced cycles of fluctuation of QH6 transcript levels were observed, and under constant dark, the cycle was delayed . However, the original diurnal pattern could be regained when the plants were returned to the normal light/dark (12 h/12 h) photoperiod . This is the first report that monoterpene biosynthesis is transcriptionally regulated in a circadian pattern.

J Biol Chem, 2002 Nov 29, 277(48), 46304 - 9 Epub 2002 Sep 10.
Subcloning, expression, purification, and characterization of recombinant human leptin-binding domain; Sandowski Y et al.; A subdomain of the human leptin receptor encoding part of the extracellular domain (amino acids 428 to 635) was subcloned, expressed in a prokaryotic host, and purified to homogeneity, as evidenced by SDS-PAGE, with over 95% monomeric protein . The purified leptin-binding domain (LBD) exhibited the predicted beta structure, was capable of binding human, ovine, and chicken leptins, and formed a stable 1:1 complex with all mammalian leptins . The binding kinetics, assayed by surface plasmon resonance methodology, showed respective k(on) and k(off) values (mean +/- S.E.) of 1.20 +/- 0.23 x 10(-5) mol(-1) s(-1) and 1.85 +/- 0.30 x 10(-3) s(-1) and a K(d) value of 1.54 x 10(-8) m . Similar results were achieved with conventional binding experiments . LBD blocked leptin-induced, but not interleukin-3-induced, proliferation of BAF/3 cells stably transfected with the long form of human leptin receptor . The modeled LBD structure and the known three-dimensional structure of human leptin were used to construct a model of 1:1 LBD.human leptin complex . Two main residues, Phe-500, located in loop L3, and Tyr-441, located in L1, are suggested to contribute to leptin binding.

Prog Biophys Mol Biol, 2002 May-Jul, 79(1-3), 77 - 133
Molecular evolution before the origin of species; Davis BK; Amino acids at conserved sites in the residue sequence of 10 ancient proteins, from 844 phylogenetically diverse sources, were used to specify their time of origin in the interval before species divergence from the last common ancestor (LCA) . The order of amino acid addition to the genetic code, based on biosynthesis path length and other molecular evidence, provided a reference for evaluating the 'code age' of each residue profile examined . Significantly earlier estimates were obtained for conserved amino acid residues in these proteins than non-conserved residues . Evidence from the primary structure of 'fossil' proteins thus corroborated the biosynthetic order of amino acid addition to the code.Low potential ferredoxin (Fdxn) had the earliest residue profile among the proteins in this study . A phylogenetic tree for 82 prokaryote Fdxn sequences was rooted midway between bacteria and archaea branches . LCA Fdxn had a 23-residue antecedent whose residue profile matched mid-expansion phase codon assignments and included an amide residue . It contained a highly acidic N-terminal region and a non-charged C-terminal region, with all four cysteine residues . This small protein apparently anchored a {4Fe-4S} cluster, ligated by C-terminal cysteines, to a positively charged mineral surface, consistent with mediating e(-) transfer in a primordial surface system before cells appeared . Its negatively charged N-terminal 'attachment site' was highly mutable during evolution of ancestral Fdxn for Bacteria and Archaea, consistent with a loss of function after cell formation . An initial glutamate to lysine substitution may link 'attachment site' removal to early post-expansion phase entry of basic amino acids to the code . As proteins evidently anchored non-charged amide residues initially, surface attachment of cofactors and other functional groups emerges as a general function of pre-cell proteins.A phylogenetic tree of 107 proteolipid (PL) helix-1 sequences from H(+)-ATPase of bacteria, archaea and eukaryotes had its root between prokaryote branches . LCA PL h1 residue profile optimally fit a late expansion phase codon array . Sequence repeats in transmembrane PL helices h1 and h2 indicated formation of the archetypal PL hairpin structure involved successive tandem duplications, initiated within the gene for an 11-residue (or 4-residue) hydrophobic peptide . Ancestral PL h1 lacked acidic residues, in a fundamental departure from the prototype pre-cell protein . By this stage, proteins with a hydrophobic domain had evolved . Its non-polar, late expansion phase residue profile point to ancestral PL being a component of an early permeable cell membrane . Other indicators of cell formation about this stage of code evolution include phospholipid biosynthesis path length, FtsZ residue profile, and late entry of basic amino acids into the genetic code.Estimates based on conserved residues in prokaryote cell septation protein, FtsZ, and proteins involved with synthesis, transcription and replication of DNA revealed FtsZ, ribonucleotide reductase, RNA polymerase core subunits and 5'-->3' flap exonuclease, FEN-1, originated soon after cells putatively evolved . While reverse transcriptase and topoisomerase I, Topo I, appeared late in the pre-divergence era, when the genetic code was essentially complete . The transition from RNA genes to a DNA genome seemingly proceeded via formation of a DNA-RNA heteroduplex . These results suggest formation of DNA awaited evolution of a catalyst with a hydrophobic domain, capable of sequestering radical bearing intermediates in its synthesis from ribonucleotide precursors . Late formation of topology altering protein, Topo I, further suggests consolidation of genes into chromosomes followed synthesis of comparatively thermostable DNA strands.

Sheng Wu Yi Xue Gong Cheng Xue Za Zhi, 2002 Jun, 19(2), 280 - 3
{Expression and purification of human papillomavirus type16 L1 protein in a prokaryotic expression system}; Bao G et al.; This study was intended to establish a method of purification of HPV16 L1 protein expressed in a prokaryotic system and to obtain the purified protein . The prokaryotic expression vector pGEX-4T-1-HPV16 L1 was constructed and transformed into E . coli BL21 cell, and induced by 1 mM IPTG to express HPV16L1 protein . The inclusion bodies were isolated and solubilized with 8 M urea . After the urea was removed by gradual dialysis, the denatured L1 protein were renatured and then were purified by affinity chromatography . The results showed that HPV16L1 protein formed inclusion bodies in bacterial expression system, suggesting that this assay can be used to purify HPV16L1 protein and hence provide a basis for studying the applications of HPV16 L1 protein.

Plant Physiol, 1997 May, 114(1), 223 - 230
Elucidation of the Biosynthesis of Eicosapentaenoic Acid in the Microalga Porphyridium cruentum (II . Studies with Radiolabeled Precursors); Khozin I et al.; In the course of the study of the biosynthesis of the fatty acid eicosapentaenoic acid (EPA) in the microalga Porphyridium cruentum, cells were pulse-labeled with various radiolabeled fatty acid precursors . Our data show that the major end products of the biosynthesis are EPA-containing galactolipids of a eukaryotic and prokaryotic nature . The prokaryotic molecular species contain EPA and arachidonic acid at the sn-1 position and C16 fatty acids, mainly 16:0, at the sn-2 positions, whereas in the eukaryotic species both positions are occupied by EPA or arachidonic acid . However, we suggest that both the eukaryotic and prokaryotic molecular species are formed in two pathways, {omega}6 and {omega}3, which involve cytoplasmic and chloroplastic lipids . In the {omega}6 pathway, cytoplasmic 18:2-phosphatidylcholine (PC) is converted to 20:4{omega}6-PC by a sequence that includes a {delta}6 desaturase, an elongation step, and a {delta}5 desaturase . In the minor {omega}3 pathway, 18:2-PC is presumably desaturated to 18:3{omega}3, which is sequentially converted by the enzymatic sequence of the {omega}6 pathway to 20:5{omega}3-PC . The products of both pathways are exported, as their diacylglycerol moieties, to the chloroplast to be galactosylated into their respective monogalactosyldiacylglycerol molecular species . The 20:4{omega}6 in both eukaryotic and prokaryotic monogalactosyldiacylglycerol can be further desaturated to EPA by a chloroplastic {delta}17 ({omega}3) desaturase.

Cell Mol Life Sci, 2002 Jul, 59(7), 1081 - 95
The galactosyltransferase family; Hennet T; Galactose is transferred via several linkages to acceptor structures by galactosyltransferase enzymes . In prokaryotes, galactose is mainly found on lipopolysaccharides and capsular polysaccharides . In eukaryotes, galactosyltransferases, which are localized in the Golgi apparatus, are involved in the formation of several classes of glycoconjugates and in lactose biosynthesis . Although they sometimes catalyze identical reactions, prokaryotic and eukaryotic galactosyltransferases share only little structural similarities . In mammals, 19 distinct galactosyltransferase enzymes have been characterized to date . These enzymes catalyze the transfer of galactose via beta1-4, beta1-3, alpha1-3 and alpha1-4 linkages . The present review focuses on the description of these mammalian galactosyltransferases.

Trends Biotechnol, 2002 Oct, 20(10), 407 - 10; discussion 410
Conservation of gene co-regulation in prokaryotes and eukaryotes; Teichmann SA et al.; Genes that are part of the same operon in prokaryotes, or have the same expression pattern in eukaryotes, are transcriptionally co-regulated . If genes are consistently co-regulated across distantly related organisms, the genes have closely associated functions . It has been shown previously that such genes have a strong tendency to belong to the same protein complex in prokaryotes, and we show by an analysis of the sequences and their expression in the yeast Saccharomyces cerevisiae and the worm Caenorhabditis elegans that this is also true for eukaryotes . Our analysis reveals that the number of conserved co-regulated genes is small in eukaryotes, as has been shown previously in prokaryotes, indicating that there are extensive variations in the gene regulatory network across organisms.

FEBS Lett, 2002 Sep 11, 527(1-3), 319 - 22
Posttranslational modification of E . coli histone-like protein H-NS and bovine histones by short-chain poly-(R)-3-hydroxybutyrate (cPHB); Reusch RN et al.; Short-chain poly-(R)-3-hydroxybutyrate (cPHB), a highly flexible, amphiphilic molecule with salt-solvating properties, is a ubiquitous constituent of prokaryotic and eukaryotic cells, wherein it is mainly conjugated to proteins . The solvating properties and cellular distribution of cPHB suggest it may be associated with proteins that bind and/or transfer DNA . Here we examine Escherichia coli protein H-NS and calf thymus histones, H1, H2A, H2B, H3, and H4, for the presence of cPHB . The proteins are related in that all bind to DNA and are implicated in the compact organization of the chromosome . The presence of cPHB in E . coli H-NS was first detected in Western blots of two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels of total cell proteins, probed with anti-cPHB IgG, and then by Western blot analysis of the purified protein . Western blot analysis of the calf thymus histones indicated that each contained cPHB . The presence of cPHB in H-NS and histones was confirmed by chemical assay . The in vivo size of conjugated cPHB could not be established due to the lack of standards and degradation of cPHB during protein purification and storage . The molecular characteristics of cPHB and its presence in histone-like and histone proteins of diverse organisms suggest it may play a role in DNA binding and/or DNA organization.

Biochem Biophys Res Commun, 2002 Sep 13, 297(1), 10 - 6
Functional properties of Kch, a prokaryotic homologue of eukaryotic potassium channels; Munsey TS et al.; To test the hypothesis that the Kch gene of Escherichia coli encodes a potassium channel, we have transformed E . coli with an expression vector containing the Kch sequence and observed the effect of over-expression of Kch on E . coli . We found that: (i) over-expression of Kch is toxic to E . coli, but the toxicity could be prevented by supplementing the growth medium with K(+), Rb(+), and NH(4)(+), but not Na(+), consistent with the properties of a potassium selective pore; (ii) Cs(+), a blocker of potassium channels, rescues the growth of Kch over-expressing cells; and (iii) when the putative pore-forming region of Kch, containing the signature sequence, was replaced with the corresponding region of the eukaryotic Shaker potassium channel, and the resultant construct expressed in E . coli, the cells became critically dependent on K(+) supply for survival . These data are consistent with the proposed function of Kch, i.e., K(+) conduction.

Environ Microbiol, 2002 Sep, 4(9), 546 - 55
Extremely productive microbial communities in shallow saline pools respond immediately to changing meteorological conditions; Kirschner AK et al.; Diel changes in bacterial and cyanobacterial numbers, as well as heterotrophic bacterial production, were examined in two shallow alkaline pools, harbouring dense populations of cyanobacteria (up to 1100 x 109 cells l-1) and bacteria (up to 500 x 109 cells l-1) . Together with the recorded bacterial production rates (925 micro gC l-1x h-1), these values are the highest reported for natural aquatic ecosystems . The investigations were performed during a fair-weather situation, and during a rapid change after a long-term fair-weather situation to thunderstorms and heavy rainfall . During fair weather, bacterial growth was significantly correlated to the diurnal light and temperature cycle . Prokaryotic abundances were fairly constant, and loss by grazing and viral lysis must have been of significant importance . During the invasion of rainy weather, the prokaryotic community showed a strong and immediate response . A significant enhancement of bacterial growth followed after rainfall, suggesting that the high salt concentrations had inhibited bacterial activity . Changes in bacterial and cyanobacterial numbers were consistent with this pattern . From comparison with the available literature, we conclude that diel changes of bacterioplankton are regulated by a complex combination of environmental factors specific for each investigated ecosystem . In the soda pools investigated, external abiotic factors were dominant on a diel scale . In larger ecosystems, such factors are much more buffered and internal biotic interactions may prevail.

Biochemistry, 2002 Sep 17, 41(37), 11071 - 9
Structure of a nitric oxide synthase heme protein from Bacillus subtilis; Pant K et al.; Eukaryotic nitric oxide synthases (NOSs) produce nitric oxide to mediate intercellular signaling and protect against pathogens . Recently, proteins homologous to mammalian NOS oxygenase domains have been found in prokaryotes and one from Bacillus subtilis (bsNOS) has been demonstrated to produce nitric oxide {Adak, S., Aulak, K . S., and Stuehr, D . J . (2002) J . Biol . Chem . 277, 16167-16171} . We present structures of bsNOS complexed with the active cofactor tetrahydrofolate and the substrate L-arginine (L-Arg) or the intermediate N(omega)-hydroxy-L-arginine (NHA) to 1.9 or 2.2 A resolution, respectively . The bsNOS structure is similar to those of the mammalian NOS oxygenase domains (mNOS(ox)) except for the absence of an N-terminal beta-hairpin hook and zinc-binding region that interact with pterin and stabilize the mNOS(ox) dimer . Changes in patterns of residue conservation between bacterial and mammalian NOSs correlate to different binding modes for pterin side chains . Residue conservation on a surface patch surrounding an exposed heme edge indicates a likely interaction site for reductase proteins in all NOSs . The heme pockets of bsNOS and mNOS(ox) recognize L-Arg and NHA similarly, although a change from Val to Ile beside the substrate guanidinium may explain the 10-20-fold slower dissociation of product NO from the bacterial enzyme . Overall, these structures suggest that bsNOS functions naturally to produce nitrogen oxides from L-Arg and NHA in a pterin-dependent manner, but that the regulation and purpose of NO production by NOS may be quite different in B . subtilis than in mammals.

J Mol Biol, 2002 Aug 30, 321(5), 785 - 96
Crystal structure and anion binding in the prokaryotic hydrogenase maturation factor HypF acylphosphatase-like domain; Rosano C et al.; {NiFe}-hydrogenases require a set of complementary and regulatory proteins for correct folding and maturation processes . One of the essential regulatory proteins, HypF (82kDa) contains a N-terminal acylphosphatase (ACT)-like domain, a sequence motif shared with enzymes catalyzing O-carbamoylation, and two zinc finger motifs similar to those found in the DnaJ chaperone . The HypF acylphosphatase domain is thought to support the conversion of carbamoylphosphate into CO and CN(-), promoting coordination of these ligands to the hydrogenase metal cluster . It has been shown recently that the HypF N-terminal domain can aggregate in vitro to yield fibrils matching those formed by proteins linked to amyloid diseases . The 1.27A resolution HypF acylphosphatase domain crystal structure (residues 1-91; R-factor 13.1%) shows a domain fold of betaalphabetabetaalphabeta topology, as observed in mammalian acylphosphatases specifically catalyzing the hydrolysis of the carboxyl-phosphate bonds in acylphosphates . The HypF N-terminal domain can be assigned to the ferredoxin structural superfamily, to which RNA-binding domains of small nuclear ribonucleoproteins and some metallochaperone proteins belong . Additionally, the HypF N-terminal domain displays an intriguing structural relationship to the recently discovered ACT domains . The structures of different HypF acylphosphatase domain complexes show a phosphate binding cradle comparable to the P-loop observed in unrelated phosphatase families . On the basis of the catalytic mechanism proposed for acylphosphatases, whereby residues Arg23 and Asn41 would support substrate orientation and the nucleophilic attack of a water molecule on the phosphate group, fine structural features of the HypF N-terminal domain putative active site region may account for the lack of acylphosphatase activity observed for the expressed domain . The crystallographic analyses here reported were undertaken to shed light on the molecular bases of inactivity, folding, misfolding and aggregation of the HypF N-terminal acylphosphatase domain.

Proc Natl Acad Sci U S A, 2002 Sep 17, 99(19), 12015 - 20 Epub 2002 Sep 06.
Physical constraints and functional characteristics of transcription factor-DNA interaction; Gerland U et al.; We study theoretical "design principles" for transcription factor (TF)-DNA interaction in bacteria, focusing particularly on the statistical interaction of the TFs with the genomic background (i.e., the genome without the target sites) . We introduce and motivate the concept of programmability, i.e., the ability to set the threshold concentration for TF binding over a wide range merely by mutating the binding sequence of a target site . This functional demand, together with physical constraints arising from the thermodynamics and kinetics of TF-DNA interaction, leads us to a narrow range of "optimal" interaction parameters . We find that this parameter set agrees well with experimental data for the interaction parameters of a few exemplary prokaryotic TFs, which indicates that TF-DNA interaction is indeed programmable . We suggest further experiments to test whether this is a general feature for a large class of TFs.

Proc Natl Acad Sci U S A, 2002 Sep 17, 99(19), 12246 - 51 Epub 2002 Sep 06.
Evolutionary analysis of Arabidopsis, cyanobacterial, and chloroplast genomes reveals plastid phylogeny and thousands of cyanobacterial genes in the nucleus; Martin W et al.; Chloroplasts were once free-living cyanobacteria that became endosymbionts, but the genomes of contemporary plastids encode only approximately 5-10% as many genes as those of their free-living cousins, indicating that many genes were either lost from plastids or transferred to the nucleus during the course of plant evolution . Previous estimates have suggested that between 800 and perhaps as many as 2,000 genes in the Arabidopsis genome might come from cyanobacteria, but genome-wide phylogenetic surveys that could provide direct estimates of this number are lacking . We compared 24,990 proteins encoded in the Arabidopsis genome to the proteins from three cyanobacterial genomes, 16 other prokaryotic reference genomes, and yeast . Of 9,368 Arabidopsis proteins sufficiently conserved for primary sequence comparison, 866 detected homologues only among cyanobacteria and 834 other branched with cyanobacterial homologues in phylogenetic trees . Extrapolating from these conserved proteins to the whole genome, the data suggest that approximately 4,500 of Arabidopsis protein-coding genes ( approximately 18% of the total) were acquired from the cyanobacterial ancestor of plastids . These proteins encompass all functional classes, and the majority of them are targeted to cell compartments other than the chloroplast . Analysis of 15 sequenced chloroplast genomes revealed 117 nuclear-encoded proteins that are also still present in at least one chloroplast genome . A phylogeny of chloroplast genomes inferred from 41 proteins and 8,303 amino acids sites indicates that at least two independent secondary endosymbiotic events have occurred involving red algae and that amino acid composition bias in chloroplast proteins strongly affects plastid genome phylogeny.

Bioinformatics, 2002 Sep, 18(9), 1266 - 7
SEGE: A database on 'intron less/single exonic' genes from eukaryotes; Sakharkar MK et al.; SUMMARY: Eukaryotes have both 'intron containing' and 'intron less' genes . Several databases are available for 'intron containing' genes in eukaryotes . In this note, we describe a database for 'intron less' genes from eukaryotes . 'Intron less' eukaryotic genes having prokaryotic architecture will help to understand gene evolution in a much simpler way unlike 'intron containing' genes . AVAILABILITY: SEGE is available at CONTACT: mmeena@ntu.edu.sg

Bioinformatics, 2002 Sep, 18(9), 1153 - 61
Genome structures embossed by oligonucleotide-stickiness; Nishigaki K et al.; MOTIVATION: An unmanageably large amount of data on genome sequences is accumulating, prompting researchers to develop new methods to analyze them . We have devised a novel method designated oligostickiness, a measure roughly proportional to the binding affinity of an oligonucleotide to a DNA of interest, in order to analyze genome sequences as a whole . RESULTS: Fifteen representative genomes such as Bacillus subtilis, Escherichia coli, Saccharomyces cerevisiae, Caenorhabditis elegans, H . sapiens and others were analyzed by this method using more than 50 probe dodecanucleotides, offering the following findings: (i) Genome sequences can be specifically featured by way of oligostickiness maps . (ii) Oligostickiness analysis, which is similar to but more informative than (G + C) content or repetitive sequence analysis, can reveal intra-genomic structures such as mosaic structures (E . coli and B . subtilis) and highly sticky/non-sticky regions of biological meanings . (iii) Some probe oligonucleotides such as dC(12) and dT(12) can be used for classifying genomes, clearly discriminating prokaryotes and eukaryotes . (iv) Based on global oligostickiness, which is the average value of the local oligostickinesses, the features of a genome could be visualized in spider web mode . The pattern of a spider web as well as a set of oligostickiness maps is highly characteristic to each genome or chromosome . Thus, we called it as chromosome texture, leading to a finding that all the chromosomes contained in a cell, so far investigated, have a common texture . AVAILABILITY: Oligostickinesses maps used in this work are available at CONTACT: koichi@fms.saitama-u.ac.jp

Extremophiles, 2002 Aug, 6(4), 275 - 81 Epub 2002 Mar 08.
L-aspartate oxidase is present in the anaerobic hyperthermophilic archaeon Pyrococcus horikoshii OT-3: characteristics and role in the de novo biosynthesis of nicotinamide adenine dinucleotide proposed by genome sequencing; Sakuraba H et al.; A gene encoding the L-aspartate oxidase homologue was identified via genome sequencing in the anaerobic hyperthermophilic archaeon Pyrococcus horikoshii OT-3 . We succeeded in expressing the encoding gene in Escherichia coli and purified the product to homogeneity . Characterization of the protein revealed that it is the most thermostable L-aspartate oxidase detected so far . In addition to the oxidase activity, the enzyme catalyzed L-aspartate dehydrogenation in the presence of an artificial electron acceptor such as phenazine methosulfate, 2,6-dichlorophenol-indophenol, and ferricyanide . L-Aspartate oxidase is known to function as the first enzyme in the de novo NAD biosynthetic pathway in prokaryotes . By a similarity search in public databases, the genes that encode the homologue of all other enzymes involved in the pathway were identified in the P . horikoshii OT-3 genome . This suggests that P . horikoshii OT-3 may use the de novo NAD biosynthetic pathway under anaerobic conditions.

Science, 2002 Sep 6, 297(5587), 1686 - 9
Identification of a DNA nonhomologous end-joining complex in bacteria; Weller GR et al.; In eukaryotic cells, double-strand breaks (DSBs) in DNA are generally repaired by the pathway of homologous recombination or by DNA nonhomologous end joining (NHEJ) . Both pathways have been highly conserved throughout eukaryotic evolution, but no equivalent NHEJ system has been identified in prokaryotes . The NHEJ pathway requires a DNA end-binding component called Ku . We have identified bacterial Ku homologs and show that these proteins retain the biochemical characteristics of the eukaryotic Ku heterodimer . Furthermore, we show that bacterial Ku specifically recruits DNA ligase to DNA ends and stimulates DNA ligation . Loss of these proteins leads to hypersensitivity to ionizing radiation in Bacillus subtilis . These data provide evidence that many bacteria possess a DNA DSB repair apparatus that shares many features with the NHEJ system of eukarya and suggest that this DNA repair pathway arose before the prokaryotic and eukaryotic lineages diverged.

J Biol Chem, 2002 Nov 22, 277(47), 45075 - 85 Epub 2002 Sep 04.
Direct in vivo screening of intrabody libraries constructed on a highly stable single-chain framework; der Maur AA et al.; Single-chain Fv antibody fragments (scFv) represent a convenient antibody format for intracellular expression in eukaryotic or prokaryotic cells . These so-called intrabodies have great potential in functional genomics as a tool to study the function of newly identified proteins in vivo, for example by binding-induced modulation of their activity or by blocking interactions with other proteins . However, the intracellular expression and activity of many single-chain Fvs are limited by their instability and folding efficiency in the reducing intracellular environment, where the highly conserved intrachain disulfide bonds do not form . In the present work, we used an in vivo selection system to isolate novel antigen-binding intrabodies . We screened two intrabody libraries carrying a randomized third hypervariable loop onto the heavy chain of a stable framework, which had been further optimized by random mutagenesis for better behavior in the selection system, and we biophysically characterized the selected variants to interpret the outcome of the selection . Our results show that single-framework intrabody libraries can be directly screened in vivo to rapidly select antigen-specific intrabodies.

J Mol Biol, 2002 Sep 6, 322(1), 53 - 64
Loopy proteins appear conserved in evolution; Liu J et al.; Over the last decade, structural biologists have unravelled many proteins that appear natively disordered . Common assumptions are that many of these proteins adopt structure through binding and that the structural flexibility enables them to adopt different functions . Here, we investigated regions of more than 70 sequence-consecutive residues that have no regular secondary structure (NORS) . Analysing 31 entirely sequenced organisms, we predicted five times as many proteins with NORS regions (loopy proteins) in eukaryotes (20%) than in prokaryotes and archaeas (4%) . Thousands of these NORS regions were over 150 residues long . The amino acid composition of NORS regions differed from that of loops in PDB . Although NORS proteins had significantly more residues in low-complexity regions than other proteins, simple cut-off thresholds for sequence bias missed most NORS regions . On average, NORS regions were evolutionarily at least as conserved as their flanking regions . Furthermore, yeast proteins with NORS regions had more protein-protein interaction partners than other proteins . Regulatory and transcription-related functions were over-represented in loopy proteins, biosynthesis and energy metabolism were under-represented . Overall, our analysis confirmed that proteins with non-regular structures appear to play important functional roles, and they may adopt as yet unknown types of protein structures.

Biochim Biophys Acta, 2002 Sep 13, 1577(2), 325 - 36
Regulation of viral transcription elongation and termination during vaccinia virus infection; Condit RC et al.; Vaccinia virus provides a useful genetic and biochemical tool for studies of the basic mechanisms of eukaryotic transcription . Vaccinia genes are transcribed in three successive gene classes during infection, early, intermediate, and late . Vaccinia transcription is regulated primarily by virus gene products not only during initiation, but also during elongation and termination . The factors and mechanisms regulating early elongation and termination differ from those regulating intermediate and late gene expression . Control of transcription elongation and termination in vaccinia virus bears some similarity to the same process in other prokaryotic and eukaryotic systems, yet features some novel mechanisms as well.

Biochim Biophys Acta, 2002 Sep 13, 1577(2), 287 - 307
Promoting elongation with transcript cleavage stimulatory factors; Fish RN et al.; Transcript elongation by RNA polymerase is a dynamic process, capable of responding to a number of intrinsic and extrinsic signals . A number of elongation factors have been identified that enhance the rate or efficiency of transcription . One such class of factors facilitates RNA polymerase transcription through blocks to elongation by stimulating the polymerase to cleave the nascent RNA transcript within the elongation complex . These cleavage factors are represented by the Gre factors from prokaryotes, and TFIIS and TFIIS-like factors found in archaea and eukaryotes . High-resolution structures of RNA polymerases and the cleavage factors in conjunction with biochemical investigations and genetic analyses have provided insights into the mechanism of action of these elongation factors . However, there are yet many unanswered questions regarding the regulation of these factors and their effects on target genes.

Biochim Biophys Acta, 2002 Sep 13, 1577(2), 191 - 207
Promoter clearance and escape in prokaryotes; Hsu LM; Promoter escape is the last stage of transcription initiation when RNA polymerase, having initiated de novo phosphodiester bond synthesis, must begin to relinquish its hold on promoter DNA and advance to downstream regions (DSRs) of the template . In vitro, this process is marked by the release of high levels of abortive transcripts at most promoters, reflecting the high instability of initial transcribing complexes (ITCs) and indicative of the existence of barriers to the escape process . The high abortive initiation level is the result of the existence of unproductive ITCs that carry out repeated initiation and abortive release without escaping the promoter . The formation of unproductive ITCs is a widespread phenomenon, but it occurs to different extent on different promoters . Quantitative analysis of promoter mutations suggests that the extent and pattern of abortive initiation and promoter escape is determined by the sequence of promoter elements, both in the promoter recognition region (PRR) and the initial transcribed sequence (ITS) . A general correlation has been found that the stronger the promoter DNA-polymerase interaction, the poorer the ability of RNA polymerase to escape the promoter . In gene regulation, promoter escape can be the rate-limiting step for transcription initiation . An increasing number of regulatory proteins are known to exert their control at this step . Examples are discussed with an emphasis on the diverse mechanisms involved . At the molecular level, the X-ray crystal structures of RNA polymerase and its various transcription complexes provide the framework for understanding the functional data on abortive initiation and promoter escape . Based on structural and biochemical evidence, a mechanism for abortive initiation and promoter escape is described.

Bioessays, 2002 Sep, 24(9), 850 - 7
The modulator is a constitutive enhancer of a developmentally regulated sea urchin histone H2A gene; Spinelli G et al.; Going back to the late 1970s and early 1980s, we trace the Xenopus oocyte microinjection experiments that led to the emergence of the concept of "modulator" . The finding that the modulator could transactivate transcription from far upstream and in either orientation suggested that a new genetic element, different from the classical prokaryotic promoter sequences, had been discovered . This particular enhancer transactivates transcription of the sea urchin early (alpha) histone H2A gene which is regulated in early sea urchin development . We summarise the data from sea urchin microinjection experiments that confirm and extend the results obtained with Xenopus oocytes . We conclude that the H2A enhancer is bipartite, is located approx . 100 bp upstream of the TATAAATA box in the H2A gene of two sea urchin species and enhances transcription when placed at a position far upstream or far downstream of the gene unless an insulator intervenes between enhancer and promoter . Evidence from microinjection experiments with sea urchin embryos suggests that the developmental control of H2A expression resides not with the enhancer, which is constitutively active, but with a striking chromatin structure with two positioned nucleosomes near the 3' end of the gene . Within this structure, there is an insulator element .

Biotechnol Bioeng, 2002 Sep 5, 79(5), 504 - 8
A memorial review of Jay Bailey's contribution in prokaryotic metabolic engineering; Hatzimanikatis V et al.; When mentioning prokaryotic metabolic engineering, most people will immediately think of Jay Bailey . Jay's contribution to this fast-growing field is evident and familiar to many . Therefore, instead of a detailed technical review, we attempt in this article to summarize his contribution and dissect reasons for his success in this area from a standpoint of one of his former students (VH) and of a colleague in the field (JCL) . This short review is by no means complete and provides only a partial view of Jay's contribution to the metabolic engineering of prokaryotes .

Mol Microbiol, 2002 Sep, 45(5), 1365 - 77
The ParB protein of Streptomyces coelicolor A3(2) recognizes a cluster of parS sequences within the origin-proximal region of the linear chromosome; Jakimowicz D et al.; The mycelial prokaryote Streptomyces coelicolor A3(2) possesses a large linear chromosome (8.67 Mb) with a centrally located origin of replication (oriC) . Recently, chromosome partitioning genes (parA and parB) and putative ParB binding sites (parS sequences) were identified in its genome . The S . coelicolor chromosome contains more parS sequences than any other bacterial chromosome characterized so far . Twenty of the 24 parS sequences are densely packed within a relatively short distance (approximately 200 kb) around oriC . A series of in vitro and in vivo experiments showed that S . coelicolor ParB protein interacts specifically with the parS sequences, albeit with a rather low affinity . Our results suggested that the binding of ParB is not only determined by the parS sequence, but also by the location of target DNA close to oriC . The unusually high number and close proximity to each other of the parS sites, together with in vivo and in vitro evidence that multiple ParB molecules may assemble along the DNA from an initial ParB-parS complex, suggest that a large DNA segment around the replication origin may form a massive nucleoprotein complex as part of the replication-partitioning cycle.

Mol Microbiol, 2002 Sep, 45(5), 1191 - 6
The dynamic microbe: green fluorescent protein brings bacteria to light; Southward CM et al.; The demonstration that the green fluorescent protein (GFP) from the jellyfish Aequorea victoria required no jellyfish-specific cofactors and could be expressed as a fluorescent protein in heterologous hosts including both prokaryotes and eukaryotes sparked the development of GFP as one of the most common reporters in use today . Over the past several years, the utility of GFP as a reporter has been optimized through the isolation and engineering of variants with increased folding rates, different in vivo stabilities and colour variants with altered excitation and emission spectral properties . One of the great utilities of GFP is as a probe for characterizing spatial and temporal dynamics of gene expression, protein localization and protein-protein interactions in living cells . The innovative application of GFP as a reporter in bacteria has made a significant contribution to microbial cell biology . This review will highlight recent studies that demonstrate the potential of GFP for real-time analysis of gene expression, protein localization and the dynamics of signalling transduction pathways through protein-protein interactions.

Nat Biotechnol, 2002 Sep, 20(9), 901 - 7 Epub 2002 Aug 19.
Macrolide-based transgene control in mammalian cells and mice; Weber W et al.; Heterologous mammalian gene regulation systems for adjustable expression of multiple transgenes are necessary for advanced human gene therapy and tissue engineering, and for sophisticated in vivo gene-function analyses, drug discovery, and biopharmaceutical manufacturing . The antibiotic-dependent interaction between the repressor (E) and operator (ETR) derived from an Escherichia coli erythromycin-resistance regulon was used to design repressible (E(OFF)) and inducible (E(ON)) mammalian gene regulation systems (E.REX) responsive to clinically licensed macrolide antibiotics (erythromycin, clarithromycin, and roxithromycin) . The E(OFF) system consists of a chimeric erythromycin-dependent transactivator (ET), constructed by fusing the prokaryotic repressor E to a eukaryotic transactivation domain that binds and activates transcription from ETR-containing synthetic eukaryotic promoters (P(ETR)) . Addition of macrolide antibiotic results in repression of transgene expression . The E(ON) system is based on E binding to artificial ETR-derived operators cloned adjacent to constitutive promoters, resulting in repression of transgene expression . In the presence of macrolides, gene expression is induced . Control of transgene expression in primary cells, cell lines, and microencapsulated human cells transplanted into mice was demonstrated using the E.REX (E(OFF) and E(ON)) systems . The macrolide-responsive E.REX technology was functionally compatible with the streptogramin (PIP-regulated and tetracycline (TET-regulated expression systems, and therefore may be combined for multiregulated multigene therapeutic interventions in mammalian cells and tissues.

Biosystems, 2002 Jun-Jul, 66(1-2), 65 - 71
Changes of the power coefficient in the 'metabolism-mass' relationship in the evolutionary process of animals; Atanasov AT et al.; The power coefficient k decreases along evolution in an allometric relationship between the oxygen consumption rate and the body mass of animals . This theoretical study investigated the role of the power coefficient k and its behavior along evolution . The animals were organized in three groups according to the values of the power coefficient k as follows: (I) from unicellular Prokaryotes to Eukaryotes; (II) from Mytilus and Annelida to Pisces; (III) from Reptilia to Mammals and Aves . At the beginning of each animal group (stage), the value of k was close to 0.9-1.0 and at the end of the stage it was close to 0.67-0.70 . Exponential sharp increase of the power coefficient k was observed during the biological transition from Protozoa to simply organized Metazoa and in the transition from Poikylothermic to Homothermic organisms (e.g . from Pisces to Reptilia) . Also, when using the periodogram regression analysis, a cyclic (periodic) pattern in this increase was observed (i.e . period T approximately 8-11 units, P<0.05) . It was postulated that the power coefficient k, as with the coefficient a, might represent the increase of complexity of animal organization within each group.

Int J Parasitol, 2002 Sep, 32(10), 1207 - 17
Biogenesis of iron-sulphur clusters in amitochondriate and apicomplexan protists; Seeber F; During the last 4 years there has been an enormous interest in the question how iron-sulphur ({Fe-S}) clusters, which are essential building blocks for life, are synthesised and assembled into apo-proteins, both in prokaryotes and in eukaryotes . The emerging picture is that the basic mechanism of this pathway has been well conserved during evolution . In yeast and probably all other eukaryotes the mitochondrion is the place where {Fe-S} clusters are synthesised, even for extramitochondrial {Fe-S} cluster-containing proteins, and a number of proteins have been functionally characterised to a certain extent within this pathway . However, almost nothing is known about this aspect in parasitic protists, although recent studies of amitochondriate protists and on the plastid-like organelle of apicomplexan parasites, the apicoplast, have started to change this . In this article I will summarise the current view of {Fe-S} cluster biogenesis in eukaryotes and discuss its implications for amitochondriate protists and for the plastid-like organelle of apicomplexan parasites.

J Biochem (Tokyo), 2002 Sep, 132(3), 433 - 9
Spectroscopic analysis of recombinant rat histidine decarboxylase; Olmo MT et al.; Mammalian histidine decarboxylases have not been characterized well owing to their low amounts in tissues and instability . We describe here the first spectroscopic characterization of a mammalian histidine decarboxylase, i.e . a recombinant version of the rat enzyme purified from transformed Escherichia coli cultures, with similar kinetic constants to those reported for mammalian histidine decarboxylases purified from native sources . We analyzed the absorption, fluorescence and circular dichroism spectra of the enzyme and its complexes with the substrate and substrate analogues . The pyridoxal-5'-phosphate-enzyme internal Schiff base is mainly in an enolimine tautomeric form, suggesting an apolar environment around the coenzyme . Michaelis complex formation leads to a polarized, ketoenamine form of the Schiff base . After transaldimination, the coenzyme-substrate Schiff base exists mainly as an unprotonated aldimine, like that observed for dopa decarboxylase . However, the coenzyme-substrate Schiff base suffers greater torsion than that observed in other L-amino acid decarboxylases, which may explain the relatively low catalytic efficiency of this enzyme . The active center is more resistant to the formation of substituted aldamines than the prokaryotic homologous enzyme and other L-amino acid decarboxylases . Characterization of the similarities and differences of mammalian histidine decarboxylase with respect to other homologous enzymes would open new perspectives for the development of new and more specific inhibitors with pharmacological potential.

Environ Toxicol, 2002, 17(4), 341 - 50
Genotoxicity of cyanobacterial extracts containing microcystins from Polish water reservoirs as determined by SOS chromotest and comet assay; Mankiewicz J et al.; Toxicity of cyanobacterial blooms, an increasing problem around the world, is connected to the increase in bloom samples containing microcystins, caused by excessive eutrophication of drinking- and recreational water reservoirs . Microcystins are the most common group of cyanobacterial hepatotoxins . In Poland they are produced mainly by the Microcystis genus . The toxicity of microcystins has been well documented, but investigation into their genotoxicity has been insufficient relative to the study of their overall toxicity . Therefore, the aim of this study was the estimation and comparison of the genotoxicity of cyanobacterial extracts with microcystins (CEMs) using the SOS chromotest (bacterial test) with Escherichia coli PQ37 and the comet assay with human lymphocytes . Cyanobacterial bloom samples were collected in the summer months from two Polish water reservoirs, one at Sulejow and one at Jeziorsko . The SOS chromotest, which used prokaryotic cells (without metabolic activation), and the comet assay, which used eukaryotic cells, both indicated the potential genotoxic effect of CEMs . Cyanobacterial extracts caused DNA damage in human lymphocytes in vitro . The maximum level of DNA damage was observed after 12 h incubation with CEMs . The bacterial test indicated a dependence of the degree of CEM genotoxicity, the composition, and the concentration of microcystins in each bloom sample examined with the time of exposure . Differences between the genotoxicity of cyanobacterial extract and the standard microcystin-LR were noticeable . This was probably caused by the interaction of different microcystin variants . The results showed that CEMs from Polish water reservoirs were genotoxic, which was reflected by the stimulation of the SOS repair system in bacterial cells (SOS chromotest) and by the damage induced in DNA in human lymphocytes (comet assay) .

Nucleic Acids Res, 2002 Sep 1, 30(17), 3643 - 52
Novel domains and orthologues of eukaryotic transcription elongation factors; Ponting CP; The passage of RNA polymerase II across eukaryotic genes is impeded by the nucleosome, an octamer of histones H2A, H2B, H3 and H4 dimers . More than a dozen factors in the yeast Saccharomyces cerevisiae are known to facilitate transcription elongation through chromatin . In order to better understand the evolution and function of these factors, their sequences have been compared with known protein, EST and DNA sequences . Elongator subcomplex components Elp4p and Elp6p are shown to be homologues of ATPases, yet with substitutions of amino acids critical for ATP hydrolysis, and novel orthologues of Elp5p are detectable in human, and other animal, sequences . The yeast CP complex is shown to contain a likely inactive homologue of M24 family metalloproteases in Spt16p/Cdc68p and a 2-fold repeat in Pob3p, the orthologue of mammalian SSRP1 . Archaeal DNA-directed RNA polymerase subunit E" is shown to be the orthologue of eukaryotic Spt4p, and Spt5p and prokaryotic NusG are shown to contain a novel 'NGN' domain . Spt6p is found to contain a domain homologous to the YqgF family of RNases, although this domain may also lack catalytic activity . These findings imply that much of the transcription elongation machinery of eukaryotes has been acquired subsequent to their divergence from prokaryotes.

Cell, 2002 Aug 23, 110(4), 407 - 13
Shared strategies in gene organization among prokaryotes and eukaryotes; Lawrence JG; Although genes in prokaryotes and eukaryotes are transcribed and translated by very different mechanisms, they may be organized in their respective chromosomes in surprisingly similar ways . Here, I examine common modes of maintaining nonrandom gene organization in both prokaryotes and eukaryotes, the different ways these organizations have likely arisen, and classes of organization that may be unique to one group or the other.

Curr Opin Genet Dev, 2002 Oct, 12(5), 512 - 8
Intramembrane proteolysis controls diverse signalling pathways throughout evolution; Urban S et al.; Regulated intramembrane proteolysis (RIP) has recently emerged as a conserved mechanism for controlling many signalling pathways in both prokaryotes and eukaryotes . Although early examples were confined to the activation of membrane-tethered transcription factors in the cell receiving the signal, recent analysis indicates that RIP also regulates the emission of factors involved in intercellular communication.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2002 Sep, 34(5), 675 - 9
{Expression, refolding and biological activity of recombinant type-I metalloproteinase acutolysin a from Agkistrodon acutus}; Xiang KJ et al.; Type-I snake venom metalloproteinase acutolysin A gene was cloned into the prokaryotic expression vector pBAD/gIIIA and the resulting recombinant plasmid pDS was obtained . By the induction with 0.02% L-(+)-arabinose, the recombinant metalloproteinase was expressed in insoluble inclusion body in E . coli TOP10 and reached up to 5%--10% of total bacterial proteins . The recombinant metalloproteinase has an additional sequence of N-terminal 22 amino acids and C-terminal 8 amino acids (housing 6 histidines), both of which derived from the vector . The purified inclusion body was solubilized by 8 mol/L urea or 6 mol/L guanidine-HCl and the denatured soluble recombinant metalloproteinase was allowed to refold in vitro . Western blotting and ELISA obviously showed that the renatured recombinant metalloproteinase possessed strong immune reactivity very closely related to natural acutolysin A . Animal experiments showed that the refolded recombinant metalloproteinase had an obvious hemorrhagic activity . Except PMSF, 1 mmol/L EDTA, 1 mmol/L EGTA, and 3 mmol/L imidazole could inhibit the hemorrhagic activity of the recombinant and the natural metalloproteinases to different extent . Based on the investigations of others and our experimental results, the hemorrhagic mechanism of snake metalloproteinases was discussed.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2002 Sep, 34(5), 671 - 4
{Preparation of polyacrylamide gel electrophoresis for human chorionic gonadotropin chimeric peptide 12 expressed in E . coli}; Zou YS et al.; In recent years, development of chimeric peptide (CP) immunogens is a trend in the vaccinological field . The CPs contain a B cell epitope(s) of target antigen and a promiscuous self - or foreign- T cell epitope(s) . However, such constructed CPs were all expressed in prokaryotic or eukaryotic systems at lower levels . To purify the human chorionic gonadotropin (hCG) CP12 expressed in E . coli at the level of about 1% of the total cell proteins, an improved method of preparative gel polyacrylamide gel electrophoresis (PAGE) was developed . The important improvement to routine preparative PAGE involves: (1) running reversed electrophoresis by rearranging the gel- carrying plate when the bromophenol blue band arrived at 1-1.5 centimeter from the bottom of the gel; (2) making a collecting trough between the gel and a dialytic membrane that was used to isolate the upper tank buffer . About 8 fractions were collected at regular intervals of 15 minutes after bromophenol blue running out of gel . And then 0.2 ml was taken from each fraction and the protein was precipitated by sequentially adding trichloroacetic acid and acetone . Each sample was dissolved in 20 microL sample buffer and analyzed and identified by SDS-PAGE and Western blotting . As a result, the hCG CP12 expression product with 95% relative homogeneity was harvested at a 50-100 microgram level after a single-step purification of this preparative PAGE, with respect to the sample which contained 3-4 mg of cell proteins.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2002 Sep, 34(5), 595 - 600
{Expression, purification and characterization of human prorelaxin-like-protein H2 in Escherichia coli}; Chen LM et al.; The cDNA gene encoding human prorelaxin H2 was subcloned from plasmid pMAL p2X-hRLX H2 into the EcoRI and BamHI sites of prokaryotic expression vector pBV220, resulting in the recombinant plasmid pBV220-hPRLX H2, which was subsequently transformed into Escherichia coli DH5arg; . The target gene was highly expressed in the form of inclusion body by thermoinduction at 42 degrees . Recombinant human Met-prorelaxin-like-protein H2 was refolded in vitro, purified by Sephadex G-75 gel filtration chromatography and reverse phase fast protein liquid chromatography . The yield was approximately 2-3 mg/L . The purified recombinant human Met-prorelaxin-like-protein H2 was shown to be a single band by 15% SDS-PAGE and gave correct amino acid composition of Met-prorelaxin . Molecular weight of the purified recombinant human Met-prorelaxin-like-protein H2 was measured to be 18 390.4 (calculated theoretical value 18 392.3) by matrix assisted laser desorption ionization time-of-flight mass spectroscopy.

Nature, 2002 Aug 29, 418(6901), 942 - 8
Open channel structure of MscL and the gating mechanism of mechanosensitive channels; Perozo E et al.; Mechanosensitive channels act as membrane-embedded mechano-electrical switches, opening a large water-filled pore in response to lipid bilayer deformations . This process is critical to the response of living organisms to direct physical stimulation, such as in touch, hearing and osmoregulation . Here, we have determined the structural rearrangements that underlie these events in the large prokaryotic mechanosensitive channel (MscL) using electron paramagnetic resonance spectroscopy and site-directed spin labelling . MscL was trapped in both the open and in an intermediate closed state by modulating bilayer morphology . Transition to the intermediate state is characterized by small movements in the first transmembrane helix (TM1) . Subsequent transitions to the open state are accompanied by massive rearrangements in both TM1 and TM2, as shown by large increases in probe dynamics, solvent accessibility and the elimination of all intersubunit spin-spin interactions . The open state is highly dynamic, supporting a water-filled pore of at least 25 A, lined mostly by TM1 . These structures suggest a plausible molecular mechanism of gating in mechanosensitive channels.

J Biol Chem, 2002 Nov 1, 277(44), 41401 - 9 Epub 2002 Aug 26.
Ribosomal proteins at the stalk region modulate functional rRNA structures in the GTPase center; Uchiumi T et al.; Replacement of the L10.L7/L12 protein complex and L11 in Escherichia coli ribosomes with the respective rat counterparts P0.P1/P2 and eukaryotic L12 causes conversion of ribosomal specificity for elongation factors from prokaryotic elongation factor (EF)-Tu/EF-G to eukaryotic EF (eEF)-1alpha/eEF-2 . Here we have investigated the effects of protein replacement on the structure and function of two rRNA domains around positions 1070 and 2660 (sarcin/ricin loop) of 23 S rRNA . Protein replacement at the 1070 region in E . coli 50 S subunits was demonstrated by chemical probing analysis . Binding of rat proteins to the 1070 region caused increased accessibility of the 2660 and 1070 regions to ligands for eukaryotic ribosomes: the ribotoxin pepocin for the 2660 region (E . coli numbering), anti-28 S autoantibody for the 1070 region, and eEF-2 for both regions . Moreover, binding of the E . coli L10.L7/L12 complex and L11 to the 1070 region was shown to be responsible for E . coli ribosomal accessibility to another ribotoxin, gypsophilin . Ribosomal proteins at the 1070 region appear to modulate the structures and functions of the 2660 and 1070 RNA regions in slightly different modes in prokaryotes and eukaryotes.

Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 2002 Jun, 16(2), 184 - 6
{Expression, purification and diagnostic application of EBV TK kinase}; Huang B et al.; BACKGROUND: To find a rapid and sensitive method for early diagnosis of nasopharyngeal carcinoma by using EBV TK kinase . METHODS: Prokaryotic expression plasmid pRSETTK was constructed . EBV TK kinase was highly expressed in E.coil BL21 (DE3) . The authors identified specificity of TK kinase by Western blot, then used purified TK kinase in ELISA to detect the IgG antibody in the serum of NPC patients . RESULTS: Specific IgG antibody against TK kinase was found in the serum of NPC patients . The specificity and sensitivity of TK kinase were both 100% in Western blot and were 98.0% and 93.4% respectively in ELISA . CONCLUSIONS: The EBV TK kinase showed high specificity and sensitivity in ELISA, therefore it can be used for early diagnosis of NPC

Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 2002 Jun, 16(2), 176 - 8
{Cloning and expression of HSV-I, II type-common antigen gD in Escherichia coli}; Li M et al.; BACKGROUND: To clone the type common antigen gD of human herpes simplex virus I, II (HSV-I, II), the authors constructed recombinant expression vector Pmal-c2/gD and induced to express the fusion protein MBP-gD . METHODS: The authors extracted HSV DNA,amplified gD gene by PCR assay and directly cloned it into prokaryotic expression vector pMAL-c2, then transformed it into E.coli DH5alpha . After proved to be correct by PCR, double enzyme digestion and sequencing, the fusion protein is induced to express by IPTG and detected by both Western blot and ELISA . RESULTS: The constructed expression vector pMAL-c2/gD can be expressed with high efficiency . The product expressed was about 35.5% of the total bacterium proteins by SDS?PAGE analysis and was found nearly 39% as soluble protein,61% as inclusion in cytoplasm . CONCLUSIONS: The authors constructed recombinant expression vector pMAL-c2/gD, the Western blotting result showed that the recombinant protein could be identified with gD specific monoclonal antibody DL6 . Therefore the protein was of natural antigenic structure of gD.

Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 2002 Jun, 16(2), 168 - 70
{Cloning and expression of simplex herpes virus ? US4 fragment}; Yi Y et al.; BACKGROUND: To get early laboratory study of type specific antigenicity of herpes simplex virus II . METHODS: PCR and prokaryotic expression technique . RESULTS: Herpes simplex virus II type specific gene fragment was expressed in E.coli and the products can be used as specific antigen for the detection of anti\HSV in the recovery sera . CONCLUSIONS: Cloning and express of HSVII type specific antigen found the basis for developing specific diagnosis methods and vaccine of HSV.

Biochem Soc Trans, 2002 Aug, 30(4), 397 - 401
14-3-3s are DNA-replication proteins; Zannis-Hadjopoulos M et al.; 14-3-3 proteins are conserved multifunctional molecules, involved in many biological processes . Several 14-3-3 isoforms were recently shown to be cruciform DNA-binding proteins, which is a new activity ascribed to the 14-3-3 family . As cruciform-binding proteins, 14-3-3 proteins are putatively involved in the regulation of DNA replication . Inverted repeat sequences that are able to extrude into cruciform structures are a common feature of replication origins in both prokaryotes and eukaryotes . The involvement of cruciform structures in the initiation of DNA replication has been demonstrated . A leading model of 14-3-3 function proposes that they facilitate critical protein-protein interactions, thus serving as a central component of a wide variety of cellular processes.

Postepy Hig Med Dosw, 2002, 56(3), 263 - 71
{Molecular basis of ion selectivity based on crystalline structure of bacterial channels}; Koprowski P et al.; The principles of ionic selectivity of the two crystallised bacterial ion channels are described . These channels are: the potassium channel KcsA, whose amino-acid sequence is homologous to the eukaryotic voltage--dependent potassium channels and the chloride channel EcClC that is a prokaryotic member of the ClC family of chloride channels . In conclusion: although the overall molecular architecture of KcsA is different from that of EcClC, the selectivity filters in both cases show similarities . They both utilise helix dipoles organised within the channel molecule in such a fashion to produce electrostatically favourable environment for anions (in the case of EcClC) or cations (in the case of KcsA).

Ann Endocrinol (Paris), 2002 Jun, 63(3), 197 - 218
{Water homeostasis in the living: molecular organization, osmoregulatory reflexes and evolution}; Acher R; Human body weight is about 70% water; 55% of the water are in cells and 45% in extracellular compartments, mainly body fluids . Each compartment has its own osmoregulatory system . The mechanisms of intracellular osmoregulation likely appeared with the cell itself, i.e . in primitive prokaryotes, some 3.8 billions years ago . Osmotic stress responses observed in present-day bacteria, yeast, plant and animals cells in culture are very similar . Variations in the cell volume entail an extension or retraction of plasma membrane that activates mechanically-gated ion channels or mechanoreceptors . Some of them have been cloned from E . coli, the nematode C . elegans, the drosophila . Osmotic stress determines an intracellular cascade of transactivations, the last transcription factor binding to a Tonicity response element (TonE) of osmoprotective genes . These genes encode enzymes synthesizing compatible osmolytes that reequilibrate the osmotic pressure . Volume and osmolality of the biological fluids (le milieu interieur) are regulated by neuroendocrine reflexes involving an afferent neural limb from baro- and osmo-receptors to hypothalamus and an efferent endocrine limb from neurosecretory cells to target cells, the hydroosmotic cells localized in osmoregulatory organs . Afferent signals trigger the biosynthesis and the processing of neurohypophyseal preprohormones in magnocellular neurons of the supraoptic and paraventricular nuclei of hypothalamus . Vasopressin in mammais and vasotocin in other vertebrates, endowed with antidiuretic and antinatriuretic properties, act on hydroosmotic cells localized in the nephron collecting duct . A specific vasopressin receptor, the V2 type receptor, located in the basolateral membrane of the principal cells, is coupled with an heterotridimeric protein Gs that activates adenylate cyclase . The cAMP product, in turn, stimulates the protein kinase A (PKA) . The latter mobilizes 5 effectors located in the apical membrane: 1) the water channel aquaporin 2; 2) the urea transporter UT1; 3) the amiloride-sensitive sodium channel ENaC; 4) the inward-rectifying potassium channel ROM-K1; 5) the chloride channel CFTR . Modulation of each effector is ensured by an A kinase anchoring protein (AKAP) . The effectors are transported from cytosol to apical membrane through transport vesicles equipped with membrane fusion proteins (such as VAMP2) that recognize specific apical membrane proteins (such as syntaxins) . Water flows into principal cells from collecting duct lumen through AQP2 and flows out to interstitium through AQP3 and AQP4 and finally reachs blood circulation through AQP1 capillaries . Vasopressin orchestrates the live effectors so as to keep water and solutes reabsorptions in balance with volaemia and osmolality set points.

J Bacteriol, 2002 Sep, 184(18), 5104 - 12
Transposition of cyanobacterium insertion element ISY100 in Escherichia coli; Urasaki A et al.; The genome of the cyanobacterium Synechocystis sp . strain PCC6803 has nine kinds of insertion sequence (IS) elements, of which ISY100 in 22 copies is the most abundant . A typical ISY100 member is 947 bp long and has imperfect terminal inverted repeat sequences . It has an open reading frame encoding a 282-amino-acid protein that appears to have partial homology with the transposase encoded by a bacterial IS, IS630, indicating that ISY100 belongs to the IS630 family . To determine whether ISY100 has transposition ability, we constructed a plasmid carrying the IPTG (isopropyl-beta-D-thiogalactopyranoside)-inducible transposase gene at one site and mini-ISY100 with the chloramphenicol resistance gene, substituted for the transposase gene of ISY100, at another site and introduced the plasmid into an Escherichia coli strain already harboring a target plasmid . Mini-ISY100 transposed to the target plasmid in the presence of IPTG at a very high frequency . Mini-ISY100 was inserted into the TA sequence and duplicated it upon transposition, as do IS630 family elements . Moreover, the mini-ISY100-carrying plasmid produced linear molecules of mini-ISY100 with the exact 3' ends of ISY100 and 5' ends lacking two nucleotides of the ISY100 sequence . No bacterial insertion elements have been shown to generate such molecules, whereas the eukaryotic Tc1/mariner family elements, Tc1 and Tc3, which transpose to the TA sequence, have . These findings suggest that ISY100 transposes to a new site through the formation of linear molecules, such as Tc1 and Tc3, by excision . Some Tc1/mariner family elements leave a footprint with an extra sequence at the site of excision . No footprints, however, were detected in the case of ISY100, suggesting that eukaryotes have a system that repairs a double strand break at the site of excision by an end-joining reaction, in which the gap is filled with a sequence of several base pairs, whereas prokaryotes do not have such a system . ISY100 transposes in E . coli, indicating that it transposes without any host factor other than the transposase encoded by itself . Therefore, it may be able to transpose in other biological systems.

Sheng Wu Gong Cheng Xue Bao, 2002 May, 18(3), 331 - 4
{Study on construct and expression of synthetic genes encoding spider dragline silk in Escherichia coli}; Li M et al.; Dragline spider silk produced from Nephilia clavipes major ampullate is a natural fibrous protein with specific mechanical properties such as high tensile strength and elasticity . Synthetic gene monomer encoding recombinant spider silk protein, based on the known repetitive protein sequence and partial cDNA sequence of dragline silk, was constructed and expressed . DNA monomer sequences were multimerized to encode high molecular weight synthetic spider silks using a "head-to-tail" construction strategy . Multimer was cloned into pET30a(+), a prokaryotic high potency expression vector, and induced with IPTG . The protein from 8-unit repeat was produced in Escherichia coli at levels up to 20 mg/L . The protein was easily purified with high recovery by using an metal ion affinity chromatography and purity was over 90% . The results of SDS-PAGE and Western blot suggested that the mass of the expression product was about 37 kD . This value and amino acid analysis were consistent with those of theoretic calculation.

Sheng Wu Gong Cheng Xue Bao, 2002 May, 18(3), 318 - 22
{Study on optimization of expression, purification, properties and biological function of recombinant human sBLyS}; Yan XM et al.; The prokaryotic expression plasmid pET-30a(+)/sBLyS was constructed and transformed into E . coli BL21 (lambda DE3) . The recombinant protein was found to be highly expressed by the plight of soluble part and inclusion body . For the sake of enhancing the proportion of the soluble part, inducement at 16 degrees C for 12 h was ascertained . The expressing product was then purified by Ni2+ affinity chromatography gel . PI of the recombinant human sBLyS(rhsBLyS) is about 7.1-7.3 and it assembles into a homotrimer . The effect of rhsBLyS on B lymphocytes by MTT method told us the B lymphocytes' proliferating capacity dose depended on concentration and also stimulating time of the rhsBLys . With rhsBLyS(2 micrograms/mL) stimulating 3 days, B lymphocytes can proliferate the most.

Protein Sci, 2002 Sep, 11(9), 2196 - 207
The beta-barrel finder (BBF) program, allowing identification of outer membrane beta-barrel proteins encoded within prokaryotic genomes; Zhai Y et al.; Many outer membrane proteins (OMPs) in Gram-negative bacteria possess known beta-barrel three-dimensional (3D) structures . These proteins, including channel-forming transmembrane porins, are diverse in sequence but exhibit common structural features . We here report computational analyses of six outer membrane proteins of known 3D structures with respect to (1) secondary structure, (2) hydropathy, and (3) amphipathicity . Using these characteristics, as well as the presence of an N-terminal targeting sequence, a program was developed allowing prediction of integral membrane beta-barrel proteins encoded within any completely sequenced prokaryotic genome . This program, termed the beta-barrel finder (BBF) program, was used to analyze the proteins encoded within the Escherichia coli genome . Out of 4290 sequences examined, 118 (2.8%) were retrieved . Of these, almost all known outer membrane proteins with established beta-barrel structures as well as many probable outer membrane proteins were identified . This program should be useful for predicting the occurrence of outer membrane proteins in bacteria with completely sequenced genomes.

Biochim Biophys Acta, 2002 Sep 2, 1592(1), 79 - 87
Insertion of proteins into the inner membrane of mitochondria: the role of the Oxa1 complex; Stuart R; The inner mitochondrial membrane harbors a large number of proteins that display a wide range of topological arrangements . The majority of these proteins are encoded in the cell's nucleus, but a few polytopic proteins, all subunits of respiratory chain complexes are encoded by the mitochondrial genome . A number of distinct sorting mechanisms exist to direct these proteins into the mitochondrial inner membrane . One of these pathways involves the export of proteins from the matrix into the inner membrane and is used by both proteins synthesized within the mitochondria, as well as by a subset of nuclear encoded proteins . Prior to embarking on the export pathway, nuclear encoded proteins using this sorting route are initially imported into the mitochondrial matrix from the cytosol, their site of synthesis . Protein export from the matrix into the inner membrane bears similarities to Sec-independent protein export in bacteria and requires the function of the Oxa1 protein . Oxa1 is a component of a general protein insertion site in yeast mitochondrial inner membrane used by both nuclear and mitochondrial DNA encoded proteins . Oxa1 is a member of the conserved Oxa1/YidC/Alb3 protein family found throughout prokaryotes throughout eukaryotes (where it is found in mitochondria and chloroplasts) . The evidence to demonstrate that the Oxa1/YidC/Alb3 protein family represents a novel evolutionarily conserved membrane insertion machinery is reviewed here.

Mol Cell, 2002 Aug, 10(2), 409 - 15
Structure of the yeast RNA polymerase II holoenzyme: Mediator conformation and polymerase interaction; Davis JA et al.; The holoenzyme formed by RNA polymerase II (RNAPII) and the Mediator complex is the target of transcriptional regulators in vivo . A three-dimensional structure of the yeast holoenzyme has been generated from electron microscopic images of single holoenzyme particles . Extensive changes in Mediator conformation required for interaction with RNAPII have been modeled by correlating the polymerase-bound and free Mediator structures . Determination of the precise orientation of the RNAPII in the holoenzyme indicates that Mediator contacts are centered on the RNAPII Rpb3/Rpb11 heterodimer, the eukaryotic homolog of the alpha(2) homodimer involved in transcription regulation in prokaryotes . Implications for the possible mechanism of transcription regulation by Mediator are discussed.

BMC Genomics . 2002 Aug 21;3(1):26.
Mouse ribonuclease III . cDNA structure, expression analysis, and chromosomal location; Fortin KR et al.; BACKGROUND: Members of the ribonuclease III superfamily of double-stranded(ds)-RNA-specific endoribonucleases participate in diverse RNA maturation and decay pathways in eukaryotic and prokaryotic cells . A human RNase III orthologue has been implicated in ribosomal RNA maturation . To better understand the structure and mechanism of mammalian RNase III and its involvement in RNA metabolism we determined the cDNA structure, chromosomal location, and expression patterns of mouse RNase III . RESULTS: The predicted mouse RNase III polypeptide contains 1373 amino acids (approximately 160 kDa) . The polypeptide exhibits a single C-terminal dsRNA-binding motif (dsRBM), tandem catalytic domains, a proline-rich region (PRR) and an RS domain . Northern analysis and RT-PCR reveal that the transcript (4487 nt) is expressed in all tissues examined, including extraembryonic tissues and the midgestation embryo . Northern analysis indicates the presence of an additional, shorter form of the transcript in testicular tissue . Fluorescent in situ hybridization demonstrates that the mouse RNase III gene maps to chromosome 15, region B, and that the human RNase III gene maps to a syntenic location on chromosome 5p13-p14 . CONCLUSIONS: The broad transcript expression pattern indicates a conserved cellular role(s) for mouse RNase III . The putative polypeptide is highly similar to human RNase III (99% amino acid sequence identity for the two catalytic domains and dsRBM), but is distinct from other eukaryotic orthologues, including Dicer, which is involved in RNA interference . The mouse RNase III gene has a chromosomal location distinct from the Dicer gene.

Mikrobiol Z, 2002 Mar-Apr, 64(2), 82 - 94
{Fructose-bisphosphatase of microorganisms}; Skrypal' IG et al.; Data from modern scientific literature concerning general characteristics of fructose-bisphosphatase--the key enzyme of microorganisms gluconeogenesis pathway have been analyzed in the survey . The conditions of fructose-bisphosphatase functioning in the cell--activation and repression, have been described . Regulation of prokaryotes enzyme activity by fructose-1.6-bisphosphate, fructose-2.6-bisphosphate, phosphoenol pyruvate, metal cation, etc., is discussed . Absolute dependence of fructose-bisphosphatase activity on cations of bivalent metals (Mg2+, Mn2+) and AMP is shown . Special attention was given to the functioning and characteristics of the enzyme in prokaryotes and eukaryotes: their similarity and differences . Data about the basic enzyme of gluconeogenesis pathway in Gram-positive and Gram-negative microorganisms, conditions of its functioning in autotrophs and heterotrophs have been generalized for the first time . Genetical and molecular biological properties of fructose-bisphosphatase in microorganisms have been considered . At the same time, the fact is established that the above enzyme has not been investigated in numerous microorganisms.

EMBO Rep, 2002 Sep, 3(9), 881 - 6 Epub 2002 Aug 16.
Conversion of omnipotent translation termination factor eRF1 into ciliate-like UGA-only unipotent eRF1; Seit-Nebi A et al.; In eukaryotic ribosomes, termination of translation is triggered by class 1 polypeptide release factor, eRF1 . In organisms with a universal code, eRF1 responds to three stop codons, whereas, in ciliates with variant codes, only one or two codon(s) remain(s) as stop signals . By mutagenesis of the Y-C-F minidomain of the N domain, we converted an omnipotent human eRF1 recognizing all three stop codons into a unipotent 'ciliate-like' UGA-only eRF1 . The conserved Cys127 located in the Y-C-F minidomain plays a critical role in stop codon recognition . The UGA-only response has also been achieved by concomitant substitutions of four other amino acids located at the Y-C-F and NIKS minidomains of eRF1 . We suggest that for eRF1 the stop codon decoding is of a non-linear (non-protein-anticodon) type and explores a combination of positive and negative determinants . We assume that stop codon recognition is profoundly different by eukaryotic and prokaryotic class 1 RFs.

J Mol Evol, 2002 Sep, 55(3), 260 - 4
Aerobiosis increases the genomic guanine plus cytosine content (GC%) in prokaryotes; Naya H et al.; The huge variation in the genomic guanine plus cytosine content (GC%) among prokaryotes has been explained by two mutually exclusive hypotheses, namely, selectionist and neutralist . The former proposals have in common the assumption that this feature is a form of adaptation to some ecological or physiological condition . On the other hand, the neutralist interpretation states that the variations are due only to different mutational biases . Since all of the traits that have been proposed by the selectionists either appeared to be limited to certain genera or were invalidated by the availability of more data, they cannot be considered as a selective force influencing the genomic GC% across all prokaryotes . In this report we show that aerobic prokaryotes display a significant increment in genomic GC% in relation to anaerobic ones . This is the first time that a link between a metabolic character and GC% has been found, independently of phylogenetic relationships and with a statistically significant amount of data.

J Biochem Mol Biol Biophys, 2002 Apr, 6(2), 117 - 21
Cloning and expression of the phosphoprotein gene of Newcastle disease virus in Escherichia coli; Kho CL et al.; The phosphoprotein (P) gene of a heat stable Newcastle disease virus (NDV) was cloned, sequenced and expressed in Escherichia coli . SDS-PAGE analysis of the recombinant P protein (395 amino acids) and a C-terminal extension derivative (424 amino acids), gave rise to two distinct protein bands with molecular masses of approximately 53-55 and 56-58 kDa, respectively, which are approximately 26-30% heavier than those calculated from the deduced amino acid sequences . The differences in molecular mass on SDS-PAGE are thought to be attributed to the acidic nature of the P protein (pI=6.27) and also the different degrees of phosphorylation in the prokaryotic cell . Amino acid sequence comparison of the P protein among the published NDV strains showed that they were highly conserved particularly at the putative phosphorylation sites.

Curr Genet, 2002 Aug, 41(5), 291 - 310 Epub 2002 Jul 18.
The gene complement for proteolysis in the cyanobacterium Synechocystis sp . PCC 6803 and Arabidopsis thaliana chloroplasts; Sokolenko A et al.; A set of 62 genes that encode the entire peptidase complement of Synechocystis sp . PCC 6803 has been identified in the genome database of that cyanobacterium . Sequence comparisons with the Arabidopsis genome uncovered the presumably homologous chloroplast components inherited from their cyanobacterial ancestor . A systematic gene disruption approach was chosen to individually inactivate, by customary transformation strategies, the majority of the cyanobacterial genes encoding peptidase subunits that are related to chloroplast enzymes . This allowed classification of the peptidases that are required for cell viability or are involved in specific stress responses . The comparative analysis between Synechocystis and Arabidopsis chloroplast peptidases showed that: (1) homologous enzymes that arose by gene duplications in cyanobacteria are functionally diverse and frequently do not complement each other, (2) the chloroplast appears to house a number of distinct peptidase polypeptide chains of cyanobacterial origin (49) which is comparable with a cyanobacterial cell (62) and (3) the peptidase complement in plastids results from a combination of the loss of some cyanobacterial peptidases and the gain or diversification of subclasses of peptidases . This reorganization in the pattern of proteolytic enzymes may reflect distinct environmental and physiological changes between prokaryotic and organellar systems.

Genome Biol . 2002 Jun 27;3(7):REVIEWS3010 . Epub 2002 Jun 27.
The 14-3-3s; Ferl RJ et al.; Multiple members of the 14-3-3 protein family have been found in all eukaryotes so far investigated, yet they are apparently absent from prokaryotes . The major native forms of 14-3-3s are homo- and hetero-dimers, the biological functions of which are to interact physically with specific client proteins and thereby effect a change in the client . As a result, 14-3-3s are involved in a vast array of processes such as the response to stress, cell-cycle control, and apoptosis, serving as adapters, activators, and repressors . There are currently 133 full-length sequences available in GenBank for this highly conserved protein family . A phylogenetic tree based on the conserved middle core region of the protein sequences shows that, in plants, the 14-3-3 family can be divided into two clearly defined groups . The core region encodes an amphipathic groove that binds the multitude of client proteins that have conserved 14-3-3-recognition sequences . The amino and carboxyl termini of 14-3-3 proteins are much more divergent than the core region and may interact with isoform-specific client proteins and/or confer specialized subcellular and tissue localization.

Dis Aquat Organ, 2002 Jul 8, 50(2), 137 - 44
Prokaryote infections in the New Zealand scallops Pecten novaezelandiae and Chlamys delicatula; Hine PM et al.; Four intracellular prokaryotes are reported from the scallops Pecten novaezelandiae Reeve, 1853 and Chlamys delicatula Hutton, 1873 . Elongated (1025 x 110 nm), irregular (390 x 200 nm), or toroidal (410 x 200 nm) mollicute-like organisms (M-LOs) occurred free in the cytoplasm in the digestive diverticular epithelial cells of both scallop species . Those in P . novaezelandiae bore osmiophilic blebs that sometimes connected the organisms together, and some had a rod-like protrusion, both of which resemble the blebs and tip structures of pathogenic mycoplasmas . The M-LOs in C . delicatula had a slightly denser core than periphery . Round M-LOs, 335 x 170 nm, occurred free in the cytoplasm of agranular haemocytes in P . novaezelandiae, without apparent harm to the host cell . In P . novaezelandiae, 2 types of highly prevalent (95 to 100%) basophilic inclusions in the branchial epithelium contained Rickettsia-like organisms (R-LOs) . Type 1 inclusions occurred in moderately hypertrophied, intensely basophilic cells, 8 to 10 microm in diameter, containing elongate intracellular R-LOs, 2000 x 500 nm . Type 2 inclusions were elongated and moderately basophilic in markedly hypertrophic branchial epithelial cells, 50 x 20 microm in diameter, containing intracellular organisms 500 x 200 nm in diameter . The possible roles of these organisms in pathogenesis is discussed.

Biochim Biophys Acta, 2002 Jun 12, 1590(1-3), 177 - 89
Toc, tic, and chloroplast protein import; Jarvis P et al.; The vast majority of chloroplast proteins are synthesized in precursor form on cytosolic ribosomes . Chloroplast precursor proteins have cleavable, N-terminal targeting signals called transit peptides . Transit peptides direct precursor proteins to the chloroplast in an organelle-specific way . They can be phosphorylated by a cytosolic protein kinase, and this leads to the formation of a cytosolic guidance complex . The guidance complex--comprising precursor, hsp70 and 14-3-3 proteins, as well as several unidentified components--docks at the outer envelope membrane . Translocation of precursor proteins across the envelope is achieved by the joint action of molecular machines called Toc (translocon at the outer envelope membrane of chloroplasts) and Tic (translocon at the inner envelope membrane of chloroplasts), respectively . The action of the Toc/Tic apparatus requires the hydrolysis of ATP and GTP at different levels, indicating energetic requirements and regulatory properties of the import process . The main subunits of the Toc and Tic complexes have been identified and characterized in vivo, in organello and in vitro . Phylogenetic evidence suggests that several translocon subunits are of cyanobacterial origin, indicating that today's import machinery was built around a prokaryotic core.

DNA Seq, 2002 Apr, 13(2), 93 - 102
A novel two-component system of Bradyrhizobium japonicum: ElmS and ElmR are encoded in diverse orientations; Muhlencoert E et al.; By completely sequencing the Bradyrhizobium japonicum insert of cosmid C27, a pair of genes was identified which was found to be highly similar to prokaryotic two-component systems . The genes of the putative regulator protein (214 aa), elmR, and the putative sensor histidine kinase (470 aa), elmS, are divergently oriented with their putative translational start sites separated by only 85 nucleotides . This new locus is located about 10 kb upstream of the sipS signal peptidase allele . The highest similarities were found to regulatory proteins of methylotrophic bacteria . Genetically defined B . japonicum mutants were tested for their growth on different monocarbon compounds and for the symbiotic interactions with soybeans . Because no obvious phenotype could be observed the functional importance of this sensor/regulator-pair remains to be determined.

Izv Akad Nauk Ser Biol, 2002 Jul-Aug, (4), 501 - 7
{On bacterial origin of mitochondria in eukaryotes in the light of current ideas of evolution of the organic world}; Kuznetsov AP et al.; The hypothesis of bacterial origin of mitochondria, which existed until the end of the 20th century, has been confirmed on the basis of the current concepts of organic world evolution in the open sea hydrosphere and original data on the entry of bacteria (prokaryotes0 in the cells of eukaryotes and their transformation into the mitochondrial mechanism of aerobic energy metabolism . This hypothesis can now be considered as a factually substantiated theory . The process of endocytosis of bacteria in the tissues of eukaryotes, which began at the onset of transition of the anaerobic state of open sea hydrosphere and land atmosphere (Early Proterozoic), is considered as the beginning of symbiotic mode of life of organisms of the Proterozoic and Postproterozoic organic world.

Plant Physiol, 2002 Aug, 129(4), 1581 - 91
Expression and molecular analysis of the Arabidopsis DXR gene encoding 1-deoxy-D-xylulose 5-phosphate reductoisomerase, the first committed enzyme of the 2-C-methyl-D-erythritol 4-phosphate pathway; Carretero-Paulet L et al.; 1-Deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) catalyzes the first committed step of the 2-C-methyl-D-erythritol 4-phosphate pathway for isoprenoid biosynthesis . In Arabidopsis, DXR is encoded by a single-copy gene . We have cloned a full-length cDNA corresponding to this gene . A comparative analysis of all plant DXR sequences known to date predicted an N-terminal transit peptide for plastids, with a conserved cleavage site, and a conserved proline-rich region at the N terminus of the mature protein, which is not present in the prokaryotic DXR homologs . We demonstrate that Arabidopsis DXR is targeted to plastids and localizes into chloroplasts of leaf cells . The presence of the proline-rich region in the mature Arabidopsis DXR was confirmed by detection with a specific antibody . A proof of the enzymatic function of this protein was obtained by complementation of an Escherichia coli mutant defective in DXR activity . The expression pattern of beta-glucuronidase, driven by the DXR promoter in Arabidopsis transgenic plants, together with the tissue distribution of DXR transcript and protein, revealed developmental and environmental regulation of the DXR gene . The expression pattern of the DXR gene parallels that of the Arabidopsis 1-deoxy-D-xylulose 5-phosphate synthase gene, but the former is slightly more restricted . These genes are expressed in most organs of the plant including roots, with higher levels in seedlings and inflorescences . The block of the 2-C-methyl-D-erythritol 4-phosphate pathway in Arabidopsis seedlings with fosmidomycin led to a rapid accumulation of DXR protein, whereas the 1-deoxy-D-xylulose 5-phosphate synthase protein level was not altered . Our results are consistent with the participation of the Arabidopsis DXR gene in the control of the 2-C-methyl-D-erythritol 4-phosphate pathway.

Microbiology, 2002 Aug, 148(Pt 8), 2405 - 12
The signal transducer P(II) and bicarbonate acquisition in Prochlorococcus marinus PCC 9511, a marine cyanobacterium naturally deficient in nitrate and nitrite assimilation; Palinska KA et al.; The amino acid sequence of the signal transducer P(II) (GlnB) of the oceanic photosynthetic prokaryote Prochlorococcus marinus strain PCC 9511 displays a typical cyanobacterial signature and is phylogenetically related to all known cyanobacterial glnB genes, but forms a distinct subclade with two other marine cyanobacteria . P(II) of P . marinus was not phosphorylated under the conditions tested, despite its highly conserved primary amino acid sequence, including the seryl residue at position 49, the site for the phosphorylation of the protein in the cyanobacterium Synechococcus PCC 7942 . Moreover, P . marinus lacks nitrate and nitrite reductase activities and does not take up nitrate and nitrite . This strain, however, expresses a low- and a high-affinity transport system for inorganic carbon (C(i); K(m,app) 240 and 4 micro M, respectively), a result consistent with the unphosphorylated form of P(II) acting as a sensor for the control of C(i) acquisition, as proposed for the cyanobacterium Synechocystis PCC 6803 . The present data are discussed in relation to the genetic information provided by the P . marinus MED4 genome sequence.

Bioinformatics, 2002 Aug, 18(8), 1143 - 4
DiffTool: building, visualizing and querying protein clusters; Chetouani F et al.; DiffTool is a resource to build and visualize protein clusters computed from a sequence database . The package provides a clustering tool to construct protein families according to sequence similarities and a web interface to query the corresponding clusters . A subtractive genome analysis tool selects protein families specific for a genome or a group of genomes . For each protein cluster, DiffTool includes access to sequences, coloured multiple alignments and phylogenetic trees . AVAILABILITY: A cluster database built from yeast and complete prokaryotic genomes is queryable at All the Perl sources are freely available to non-profit organizations upon request.

Trends Genet, 2002 Sep, 18(9), 472 - 9
Genome trees and the tree of life; Wolf YI et al.; Genome comparisons indicate that horizontal gene transfer and differential gene loss are major evolutionary phenomena that, at least in prokaryotes, involve a large fraction, if not the majority, of genes . The extent of these events casts doubt on the feasibility of constructing a 'Tree of Life', because the trees for different genes often tell different stories . However, alternative approaches to tree construction that attempt to determine tree topology on the basis of comparisons of complete gene sets seem to reveal a phylogenetic signal that supports the three-domain evolutionary scenario and suggests the possibility of delineation of previously undetected major clades of prokaryotes . If the validity of these whole-genome approaches to tree building is confirmed by analyses of numerous new genomes, which are currently being sequenced at an increasing rate, it would seem that the concept of a universal 'species' tree is still appropriate . However, this tree should be reinterpreted as a prevailing trend in the evolution of genome-scale gene sets rather than as a complete picture of evolution.

Biochemistry, 2002 Aug 20, 41(33), 10499 - 509
Stereospecificity of aminoglycoside-ribosomal interactions; Ryu do H et al.; Aminoglycoside antibiotics bind to the A-site decoding region of bacterial rRNA causing mistranslation and/or premature message termination . Aminoglycoside binding to A-site RNA decoding region constructs is established here to be only weakly stereospecific . Mirror-image prokaryotic A-site decoding region constructs were prepared in the natural D-series and the enantiomeric L-series and tested for binding to a series of aminoglycosides . In general, aminoglycosides bind to the D-series decoding region constructs with 2-3-fold higher affinities than they bind to the enantiomeric L-series . Moreover, L-neamine, the enantiomer of naturally occurring D-neamine, was prepared and shown to bind approximately 2-fold more weakly than D-neamine to the natural series decoding region construct, a result consistent with weakly stereospecific binding . The binding of naturally occurring D-neamine and its synthetic L-enantiomer was further evaluated with respect to binding to prokaryotic and eukaryotic ribosomes . Here, weak stereospecifcity was again observed with L-neamine being the more potent binder by a factor of approximately 2 . However, on a functional level, unnatural L-neamine proved to inhibit in vitro translation with significantly lower potency (approximately 5-fold) than D-neamine . In addition, both L- and D-neamine are bacteriocidal toward Gram-(-) bacteria . L-Neamine inhibits the growth of E . coli and P . aeruginosa with 8- and 3-fold higher MIC than D-neamine . Interestingly, L-neamine also inhibits the growth of aminoglycoside-resistant E . coli, which expresses a kinase able to phosphorylate and detoxify aminoglycosides of the D-series . These observations suggest that mirror-image aminoglycosides may avoid certain forms of enzyme-mediated resistance.

Philos Trans R Soc Lond B Biol Sci, 2002 Jul 29, 357(1423), 917 - 25
Molecular basis of cold adaptation; D'Amico S et al.; Cold-adapted, or psychrophilic, organisms are able to thrive at low temperatures in permanently cold environments, which in fact characterize the greatest proportion of our planet . Psychrophiles include both prokaryotic and eukaryotic organisms and thus represent a significant proportion of the living world . These organisms produce cold-evolved enzymes that are partially able to cope with the reduction in chemical reaction rates induced by low temperatures . As a rule, cold-active enzymes display a high catalytic efficiency, associated however, with a low thermal stability . In most cases, the adaptation to cold is achieved through a reduction in the activation energy that possibly originates from an increased flexibility of either a selected area or of the overall protein structure . This enhanced plasticity seems in turn to be induced by the weak thermal stability of psychrophilic enzymes . The adaptation strategies are beginning to be understood thanks to recent advances in the elucidation of the molecular characteristics of cold-adapted enzymes derived from X-ray crystallography, protein engineering and biophysical methods . Psychrophilic organisms and their enzymes have, in recent years, increasingly attracted the attention of the scientific community due to their peculiar properties that render them particularly useful in investigating the possible relationship existing between stability, flexibility and specific activity and as valuable tools for biotechnological purposes.

Proc Natl Acad Sci U S A, 2002 Aug 20, 99(17), 11501 - 6 Epub 2002 Aug 08.
Cell and chloroplast division requires ARTEMIS; Fulgosi H et al.; Chloroplasts are endosymbiotic organelles of cyanobacterial origin . It seems reasonable to assume that cell division and organelle division still share general principles, as shown for the FtsZ proteins . However, further components involved in this process are largely unknown . Here we describe ARTEMIS, a nuclear-encoded protein of chloroplast inner envelope membranes that is required for organelle division . ARTEMIS consists of three distinct modules: an N-terminal receptor-like region, a centrally positioned glycine-rich stretch containing a nucleoside triphosphate-binding site, and a C-terminal YidC/Oxa1p/Alb3 protein translocase-like domain . Analysis of Arabidopsis En-1 transposon mutants as well as ARTEMIS antisense plants revealed chloroplasts arrested in the late stages of division . Chloroplasts showed clearly separated and distinct multiple thylakoid systems, whereas the final organelle fission remained unaccomplished . Inactivation of a cyanobacterial gene with sequence similarity to the YidC/Oxa1p/Alb3-like domain of ARTEMIS resulted in aberrant cell division, which could be rescued by the Arabidopsis protein . ARTEMIS represents a so-far-unrecognized link between prokaryotic cell fission and chloroplast division.

J Bacteriol, 2002 Sep, 184(17), 4891 - 905
Imbroglios of viral taxonomy: genetic exchange and failings of phenetic approaches; Lawrence JG et al.; The practice of classifying organisms into hierarchical groups originated with Aristotle and was codified into nearly immutable biological law by Linnaeus . The heart of taxonomy is the biological species, which forms the foundation for higher levels of classification . Whereas species have long been established among sexual eukaryotes, achieving a meaningful species concept for prokaryotes has been an onerous task and has proven exceedingly difficult for describing viruses and bacteriophages . Moreover, the assembly of viral "species" into higher-order taxonomic groupings has been even more tenuous, since these groupings were based initially on limited numbers of morphological features and more recently on overall genomic similarities . The wealth of nucleotide sequence information that catalyzed a revolution in the taxonomy of free-living organisms necessitates a reevaluation of the concept of viral species, genera, families, and higher levels of classification . Just as microbiologists discarded dubious morphological traits in favor of more accurate molecular yardsticks of evolutionary change, virologists can gain new insight into viral evolution through the rigorous analyses afforded by the molecular phylogenetics of viral genes . For bacteriophages, such dissections of genomic sequences reveal fundamental flaws in the Linnaean paradigm that necessitate a new view of viral evolution, classification, and taxonomy.

Bioinformatics, 2002, 18 Suppl 1, S329 - 36
A powerful non-homology method for the prediction of operons in prokaryotes; Moreno-Hagelsieb G et al.; MOTIVATION: The prediction of the transcription unit organization of genomes is an important clue in the inference of functional relationships of genes, the interpretation and evaluation of transcriptome experiments, and the overall inference of the regulatory networks governing the expression of genes in response to the environment . Though several methods have been devised to predict operons, most need a high characterization of the genome analysed . Log-likelihoods derived from inter-genic distance distributions work surprisingly well to predict operons in Escherichia coli and are available for any genome as soon as the gene sets are predicted . RESULTS: Here we provide evidence that the very same method is applicable to any prokaryotic genome . First, the method has the same efficiency when evaluated using a collection of experimentally known operons of Bacillus subtilis . Second, operons among most if not all prokaryotes seem to have the same tendencies to keep short distances between their genes, the most frequent distances being the overlaps of four and one base pairs . The universality of this structural feature allows us to predict the organization of transcription units in all prokaryotes . Third, predicted operons contain a higher proportion of genes with related phylogenetic profiles and conservation of adjacency than predicted borders of transcription units.

Cell Mol Life Sci, 2002 Jun, 59(6), 1024 - 41
The thioredoxin system of Plasmodium falciparum and other parasites; Rahlfs S et al.; Antioxidant defence plays a crucial role in rapidly growing and multiplying organisms, including parasites and tumor cells . Apart from reactive oxygen species (ROS) produced in endogenous reactions, parasites are usually exposed to high ROS concentrations imposed by the host immune system . The glutathione and thioredoxin systems represent the two major antioxidant defence lines in most eukaryotes and prokaryotes . Trypanosomatids, however, are characterized by their unique trypanothione system . These systems are NADPH-dependent and based on the catalytic activity of the flavoenzymes glutathione reductase, trypanothione reductase and thioredoxin reductase (TrxR), respectively . TrxR reduces the 12-kDa protein thioredoxin (Trx), which in turn provides elcctrons to ribonucleotide reductase, thioredoxin peroxidases (TPxs), certain transcription factors and other target molecules . Comparing the thioredoxin systems of different parasites and their respective host cells enhances our understanding of parasite biology and evolution, of parasite-host interactions and mechanisms of drug resistance . It furthermore opens avenues for the development of novel antiparasitic compounds . Here we review the current knowledge on the Trx systems of eukaryotic parasites, finally focusing on the malarial parasite Plasmodium falciparum.

Theor Popul Biol, 2002 Jun, 61(4), 489 - 95
Horizontal gene transfer in microbial genome evolution; Jain R et al.; Horizontal gene transfer is the collective name for processes that permit the exchange of DNA among organisms of different species . Only recently has it been recognized as a significant contribution to inter-organismal gene exchange . Traditionally, it was thought that microorganisms evolved clonally, passing genes from mother to daughter cells with little or no exchange of DNA among diverse species . Studies of microbial genomes, however, have shown that genomes contain genes that are closely related to a number of different prokaryotes, sometimes to phylogenetically very distantly related ones . (Doolittle et al., 1990, J . Mol . Evol . 31, 383-388; Karlin et al., 1997, J . Bacteriol . 179, 3899-3913; Karlin et al., 1998, Annu . Rev . Genet . 32, 185-225; Lawrence and Ochman, 1998, Proc . Natl . Acad . Sci . USA 95, 9413-9417; Rivera et al., 1998, Proc . Natl . Acad . Sci . USA 95, 6239-6244; Campbell, 2000, Theor . Popul . Biol . 57 71-77; Doolittle, 2000, Sci . Am . 282, 90-95; Ochman and Jones, 2000, Embo . J . 19, 6637-6643; Boucher et al . 2001, Curr . Opin., Microbiol . 4, 285-289; Wang et al., 2001, Mol . Biol . Evol . 18, 792-800) . Whereas prokaryotic and eukaryotic evolution was once reconstructed from a single 16S ribosomal RNA (rRNA) gene, the analysis of complete genomes is beginning to yield a different picture of microbial evolution, one that is wrought with the lateral movement of genes across vast phylogenetic distances . (Lane et al., 1988, Methods Enzymol . 167, 138-144; Lake and Rivera, 1996, Proc . Natl . Acad . Sci . USA 91, 2880-2881; Lake et al., 1999, Science 283, 2027-2028) .

Theor Popul Biol, 2002 Jun, 61(4), 409 - 22
Evolution of the Archaea; Forterre P et al.; Archaea, members of the third domain of life, are bacterial-looking prokaryotes that harbour many unique genotypic and phenotypic properties, testifying for their peculiar evolutionary status . The archaeal ancestor was probably a hyperthermophilic anaerobe . Two archaeal phyla are presently recognized, the Euryarchaeota and the Crenarchaeota . Methanogenesis was the main invention that occurred in the euryarchaeal phylum and is now shared by several archaeal groups . Adaptation to aerobic conditions occurred several times independently in both Euryarchaeota and Crenarchaeota . Recently, many new groups of Archaea that have not yet been cultured have been detected by PCR amplification of 16S ribosomal RNA from environmental samples . The phenotypic and genotypic characterization of these new groups is now a top priority for further studies on archaeal evolution .

Trends Plant Sci, 2002 Aug, 7(8), 332 - 4
Recent breakthroughs in the study of salicylic acid biosynthesis; Metraux JP; Salicylic acid is an important regulator of induced plant resistance to pathogens . Consequently, the biosynthesis of salicylic acid and its regulation has received a lot of attention . Salicylic acid can be made from phenylalanine via cinnamic and benzoic acid . Recently, genetic studies in Arabidopsis have shown that salicylic acid is made in the chloroplast from isochorismate, a pathway that is known to operate in prokaryotes.

Plant J, 2002 Aug, 31(3), 269 - 77
The topological specificity factor AtMinE1 is essential for correct plastid division site placement in Arabidopsis; Maple J et al.; In plant cells, plastids divide by binary fission involving a complex pathway of events . Although there are clear similarities between bacterial and plastid division, limited information exists regarding the mechanism of plastid division in higher plants . Here we demonstrate that AtMinE1, an Arabidopsis homologue of the bacterial MinE topological specificity factor, is an essential integral component of the plastid division machinery . In prokaryotes MinE imparts topological specificity during cell division by blocking division apparatus assembly at sites other than midcell . We demonstrate that overexpression of AtMinE1 in E . coli results in loss of topological specificity and minicell formation suggesting evolutionary conservation of MinE mode of action . We further show that AtMinE1 can indeed act as a topological specificity factor during plastid division revealing that AtMinE1 overexpression in Arabidopsis seedlings results in division site misplacement giving rise to multiple constrictions along the length of plastids . In agreement with cell division studies in bacteria, AtMinE1 and AtMinD1 show distinct intraplastidic localisation patterns suggestive of dynamic localisation behaviour . Taken together our findings demonstrate that AtMinE1 is an evolutionary conserved topological specificity factor, most probably acting in concert with AtMinD1, required for correct plastid division in Arabidopsis.

Biosci Biotechnol Biochem, 2002 Jun, 66(6), 1431 - 4
Identification of a 22-kDa protein required for the degradation of Selenomonas ruminantium lysine decarboxylase by ATP-dependent protease; Yamaguchi Y et al.; In Selenomonas ruminantium, a strictly anaerobic, Gram-negative bacterium isolated from sheep rumen, a rapid degradation of lysine decarboxylase (LDC) occurred on entry into the stationary phase of cell growth . Here, we identified a 22-kDa protein as a stimulating factor for the degradation of LDC, which was catalyzed by ATP-dependent protease(s) in S . ruminantium . The purified 22-kDa protein preparation itself had no degradation activity towards LDC but it was required for the degradation of LDC by ATP-dependent proteases in a cell-free system . The 22-kDa protein had similar biochemical and biophysical characteristics to those of antizyme, the regulator for the degradation of mammalian ODC, which had been reported only in mammalian cells . From the sequencing data of the N-terminal 30 amino acid residues of the 22-kDa protein preparation, 22-kDa protein was found to be a new protein which was distinguished from antizyme . This is the first report of the presence of an antizyme-like regulator protein in a prokaryote.

Trends Biochem Sci, 2002 Aug, 27(8), 384 - 6
SWIM, a novel Zn-chelating domain present in bacteria, archaea and eukaryotes; Makarova KS et al.; A previously undetected domain with a CxCx(n)CxH pattern of predicted zinc-chelating residues was identified in a variety of prokaryotic and eukaryotic proteins . These include bacterial ATPases of the SWI2/SNF2 family, plant MuDR transposases and transposase-derived Far1 nuclear proteins, and vertebrate MEK kinase-1 . This domain was designated SWIM after SWI2/SNF2 and MuDR, and is predicted to have DNA-binding and protein-protein interaction functions in different contexts.

Biochim Biophys Acta, 2002 Aug 19, 1577(1), 1 - 9
Small heat shock proteins and stress tolerance in plants; Sun W et al.; Small heat shock proteins (sHsps) are produced ubiquitously in prokaryotic and eukaryotic cells upon heat . The special importance of sHsps in plants is suggested by unusual abundance and diversity . Six classes of sHsps have been identified in plants based on their intracellular localization and sequence relatedness . In addition to heat stress, plant sHsps are also produced under other stress conditions and at certain developmental stages . Induction of sHsp gene expression and protein accumulation upon environmental stresses point to the hypothesis that these proteins play an important role in stress tolerance . The function of sHsps as molecular chaperones is supported by in vitro and in vivo assays . This review summarizes recent knowledge about plant sHsp gene expression, protein structure and functions.

Life Sci, 2002 Aug 30, 71(15), 1771 - 8
Transfer of anti-TFAR19 monoclonal antibody into HeLa cells by in situ electroporation can inhibit the apoptosis; Rui M et al.; Electroporation has been successfully used for the introduction of DNA, RNA, oligonucleotide and protein into eukaryotic and prokaryotic cells for the transformation and expression of various gene products . TFAR19 (TF-1 apoptosis-related gene 19), also designated PDCD5 (Programmed Cell Death 5), is cloned as an increased expression gene during the apoptotic process of TF-1 cell induced by cytokine withdrawal . It facilitates rather than induces apoptosis in different cell lines . To explore its molecular mechanism, we successfully transferred the anti-TFAR19 monoclonal antibody into HeLa cells by in situ electroporation and observed the apoptosis process of HeLa cells induced by etoposide with flow cytometry . We demonstrate that the introduction of anti-TFAR19 antibody can suppress the apoptosis accelerating effect of TFAR19 in its natural environment . This study shows that TFAR19 may be a critical factor for apoptosis; and transfer of monoclonal antibody into mammalian cells by in situ electroporation is a useful method to study the function of endogenous factors.

Chin Med J (Engl), 2002 Jul, 115(7), 1053 - 7
Recombinant human B7.2 IgV-like domain expressed in bacteria maintains its co-stimulatory activity in vitro; Yan X et al.; OBJECTIVE: To investigate which of the two immunoglobulin (Ig)-like domains, the immunoglobulin variable region homologous domain IgV (hB7.2 IgV) and the immunoglobulin constant region homologous domain IgC (hB7.2 IgC) on the human B7.2 molecule contains receptor binding sites, and to evaluate whether the B7.2 protein expressed in bacteria has biological activity in vitro . METHODS: Three fragments of hB7.2 IgV,hB7.2 IgC and the complete extracellular region of human B7.2 containing both the IgV and IgC domains,hB7.2 Ig (V+C), were amplified by PCR and subcloned into pGEM-Teasy . Three recombinants,pGEX-4T-3-hB7.2 IgV,pGEX-4T-3-hB7.2 IgC and pGEX-4T-3-hB7.2 Ig (V+C), were generated by cloning the fragments into a prokaryote expression plasmid (pGEX-4T-3) and transformed into the host strain E . coli DH5alpha . The relevant target fusion proteins consisting of GST and hB7.2 IgV,hB7.2 IgC and hB7.2 Ig (V+C), were identified by SDS-PAGE and Western blotting . With the presence of the first signal imitated by anti-CD3 antibody, T cell activation was observed by exposing purified T lymphocytes to each soluble form of the three bacterially-produced human B7.2 fusion proteins by {(3)H}-TdR incorporation . RESULTS: Three recombinant fusion proteins of human B7.2, GST-hB7.2 IgV, GST-hB7.2 IgC and GST-hB7.2 Ig (V+C) were produced and detected in inclusion body form from engineered bacteria . With the first signal present,T lymphocytes proliferated when co-stimulated by bacterially-produced either GST-hB7.2 Ig (V+C) or GST-hB7.2 IgV fusion proteins, but not by GST-hB7.2 IgC . CONCLUSIONS: Functional human B7.2 fusion protein can be produced in bacteria . The IgV-like domain of human B7.2 is sufficient for B7.2 to interact with its counter-receptors and co-stimulate T lymphocytes.

Appl Environ Microbiol, 2002 Aug, 68(8), 3878 - 85
High-throughput methods for culturing microorganisms in very-low-nutrient media yield diverse new marine isolates; Connon SA et al.; Microbial diversity studies based on the cloning and sequencing of DNA from nature support the conclusion that only a fraction of the microbial diversity is currently represented in culture collections . Out of over 40 known prokaryotic phyla, only half have cultured representatives . In an effort to culture the uncultured phylotypes from oligotrophic marine ecosystems, we developed high-throughput culturing procedures that utilize the concept of extinction culturing to isolate cultures in small volumes of low-nutrient media . In these experiments, marine bacteria were isolated and cultivated at in situ substrate concentrations-typically 3 orders of magnitude less than common laboratory media . Microtiter plates and a newly developed procedure for making cell arrays were employed to raise the throughput rate and lower detection sensitivity, permitting cell enumeration from 200-microl aliquots of cultures with densities as low as 10(3) cells/ml . Approximately 2,500 extinction cultures from 11 separate samplings of marine bacterioplankton were screened over the course of 3 years . Up to 14% of the cells collected from coastal seawater were cultured by this method, which was 14- to 1,400-fold higher than the numbers obtained by traditional microbiological culturing techniques . Among the microorganisms cultured were four unique cell lineages that belong to previously uncultured or undescribed marine Proteobacteria clades known from environmental gene cloning studies . These cultures are related to the clades SAR11 (alpha subclass), OM43 (beta subclass), SAR92 (gamma subclass), and OM60/OM241 (gamma subclass) . This method proved successful for the cultivation of previously uncultured marine bacterioplankton that have consistently been found in marine clone libraries.

Biochemistry, 2002 Aug 6, 41(31), 9765 - 75
Structural characterization of distinct alpha3N and alpha5 metal sites in the cyanobacterial zinc sensor SmtB; VanZile ML et al.; SmtB is required for Synechococcus to effect a response to toxic concentrations of Zn(II) and other heavy metals . Direct binding of inducing metal ions to SmtB transcriptionally derepresses the expression of SmtA, a prokaryotic class II metallothionein . Homodimeric SmtB binds one Zn(II) or Co(II) per monomer in a cysteine thiolate-containing site in a tetrahedral coordination geometry {VanZile, M . L., et al . (2000) Biochemistry 39, 11818-11829} . In this report, characterization of a set of cysteine substitution mutants of SmtB reveals that SmtB homodimer binds Zn(II) or Co(II) in one of two mutually exclusive metal binding sites, termed alpha3N and alpha5, with very high equilibrium affinities . Both sites are characterized by similar affinities for Co(II) (K(Co) approximately equal to 2-5 x 10(9) M(-1)), while the Zn(II) affinities are at least 20-fold different (K(Zn)(alpha)(3N) > or = 10(13) M(-1); K(Zn)(alpha)(5) approximately equal to 5 x 10(11) M(-1)) . Co(II) bound exclusively at the alpha5 sites is capable of rapid equilibration between the alpha3N and alpha5 sites upon reduction of the mixed disulfides in S-methylated SmtB . These results suggest that the alpha3N or alpha5 metal sites might play distinct roles in this Zn(II)-sensing protein, systematically investigated in the following paper {VanZile, M . L., Chen, X., and Giedroc, D . P . (2002) Biochemistry 41, 9776-9786} . Since both the alpha3N and alpha5 sites are present in many members of the SmtB/ArsR family of metal sensor proteins, the presence of these two metal binding sites may explain some of the functional diversity in metal responses across this family of proteins.

EMBO J, 2002 Aug 1, 21(15), 4183 - 95
Co-operation and communication between the human maintenance and de novo DNA (cytosine-5) methyltransferases; Kim GD et al.; Three different families of DNA (cytosine-5) methyltransferases, DNMT1, DUMT2, DNMT3a and DNMT3b, participate in establishing and maintaining genomic methylation patterns during mammalian development . These enzymes have a large N-terminal domain fused to a catalytic domain . The catalytic domain is homologous to prokaryotic (cytosine-5) methyltransferases and contains the catalytic PC dipeptide, while the N-terminus acts as a transcriptional repressor by recruiting several chromatin remodeling proteins . Here, we show that the human de novo enzymes hDNMT3a and hDNMT3b form complexes with the major maintenance enzyme hDNMT1 . Antibodies against hDNMT1 pull down both the de novo enzymes . Furthermore, the N-termini of the enzymes are involved in protein-protein interactions . Immunocytochemical staining revealed mostly nuclear co-localization of the fusion proteins, with the exception of hDNMT3a, which is found either exclusively in cytoplasm or in both nucleus and cytoplasm . Pre-methylated substrate DNAs exhibited differential methylation by de novo and maintenance enzymes . In vivo co-expression of hDNMT1 and hDNMT3a or hDNMT3b leads to methylation spreading in the genome, suggesting co-operation between de novo and maintenance enzymes during DNA methylation.

J Autoimmun, 2002 Jun, 18(4), 299 - 309
A highly sensitive and simple assay for the detection of circulating autoantibodies against full-length bullous pemphigoid antigen 180; Schmidt E et al.; Bullous pemphigoid antigen 180 (BP180) is the target of autoantibodies in various subepidermal blistering diseases . The most common one is bullous pemphigoid (BP) . The pathological importance of anti-BP180 antibodies has been demonstrated in a passive transfer mouse model . However, sensitive assays for routinely detecting circulating antibodies directed against both intra- and extracellular domains of BP180 are only available in specialized laboratories . In addition, most current assays use prokaryotic recombinant fragments of BP180 that lack conformation-dependent epitopes . A simple and very sensitive immunofluorescence (IF) assay based on eukaryotic cells is described here . Sf21 insect cells were transfected with full-length (FL) BP180 . As revealed by FACS and confocal laser scanning microscopy the protein was expressed as type II transmembrane protein as in human keratinocytes . By testing serial dilutions of BP180-specific mouse monoclonal antibodies, the eukaryotic IF assay was demonstrated to be more sensitive compared to conventional assays including (1) indirect IF microscopy of human salt-split skin, (2) Western blotting (WB) of the keratinocyte-derived BP180 ectodomain, (3) WB of recombinant BP180 NC16A, and (4) WB of FL-BP180 extracted from Sf21 insect cells . When applied to sera from patients with BP (n = 65), pemphigoid gestationis (n = 16), and cicatricial pemphigoid (n = 7), the novel assay revealed that 58 (89%), 13 (81%), and 6 (84%), respectively, were positive . In contrast, all control sera (pemphigus, n = 20; epidermolysis bullosa acquisita, n = 5; anti-laminin 5 cicatricial pemphigoid, n = 5; systemic lupus erythematosus, n = 5; atopic dermatitis, n = 7; contact dermatitis, n = 3; normal human sera, n = 30) were negative indicating that the assay is highly specific . In addition, reactivity of the assay was conserved to a large extent when the cells had been stored at -20 degrees C for 3 months . Thus, this assay meets the demands of a simple and effective diagnostic tool for detecting circulating antibodies against FL-BP180 and may also be used in laboratories without access to molecular biological technology.

J Biomol Struct Dyn, 2002 Aug, 20(1), 127 - 34
Sequence-dependent flexibility in promoter sequences; Tsai L et al.; The non-neighbor interactions between base-pairs were taken into account to calculate the angular parameters (Omega, rho and tau) describing the orientation of successive base-pair planes and the translation parameters (D(y)) along the long axis of base-pair steps for 36 independent tetramers . A statistical mechanical model was proposed to predict the DNA flexibility that is mainly related to the thermal fluctuations at individual base-pair steps . The DNA flexibility can be described by the root-mean-square deviation of the end-to-end distance of DNA helical structure . The present model was then used to investigate the extreme flexible pattern in prokaryotic and eukaryotic promoter sequences . The results demonstrated several extreme flexible regions related to functionally important elements exist both in prokaryotic promoters and in eukaryotic promoters, DNA flexibility and AT content are highly correlated . The probabilities finding flexibility pattern in promoter sequences were also estimated statistically . The biological implications were discussed briefly.

Comput Chem, 2002 Jul, 26(5), 531 - 41
Bias of purine stretches in sequenced chromosomes; Ussery D et al.; We examined more than 700 DNA sequences (full length chromosomes and plasmids) for stretches of purines (R) or pyrimidines (Y) and alternating YR stretches; such regions will likely adopt structures which are different from the canonical B-form . Since one turn of the DNA helix is roughly 10 bp, we measured the fraction of each genome which contains purine (or pyrimidine) tracts of lengths of 10 bp or longer (hereafter referred to as 'purine tracts'), as well as stretches of alternating pyrimidines/purine (pyr/pur tracts') of the same length . Using this criteria, a random sequence would be expected to contain 1.0% of purine tracts and also 1.0% of the alternating pyr/pur tracts . In the vast majority of cases, there are more purine tracts than would be expected from a random sequence, with an average of 3.5%, significantly larger than the expectation value . The fraction of the chromosomes containing pyr/pur tracts was slightly less than expected, with an average of 0.8% . One of the most surprising findings is a clear difference in the length distributions of the regions studied between prokaryotes and eukaryotes . Whereas short-range correlations can explain the length distributions in prokaryotes, in eukaryotes there is an abundance of long stretches of purines or alternating purine/pyrimidine tracts, which cannot be explained in this way; these sequences are likely to play an important role in eukaryotic chromosome organisation.

Annu Rev Microbiol, 2002, 56, 211 - 36 Epub 2002 Jan 30.
Microbial communities and their interactions in soil and rhizosphere ecosystems; Kent AD et al.; Since the first estimate of prokaryotic abundance in soil was published, researchers have attempted to assess the abundance and distribution of species and relate this information on community structure to ecosystem function . Culture-based methods were found to be inadequate to the task, and as a consequence a number of culture-independent approaches have been applied to the study of microbial diversity in soil . Applications of various culture-independent methods to descriptions of soil and rhizosphere microbial communities are reviewed . Culture-independent analyses have been used to catalog the species present in various environmental samples and also to assess the impact of human activity and interactions with plants or other microbes on natural microbial communities . Recent work has investigated the linkage of specific organisms to ecosystem function . Prospects for increased understanding of the ecological significance of particular populations through the use of genomics and microarrays are discussed.

Annu Rev Microbiol, 2002, 56, 237 - 61 Epub 2002 Jan 30.
Transition metal transport in yeast; Van Ho A et al.; All eukaryotes and most prokaryotes require transition metals . In recent years there has been an enormous advance in our understanding of how these metals are transported across the plasma membrane . Much of this understanding has resulted from studies on the budding yeast Saccharomyces cerevisiae . A variety of genetic and biochemical approaches have led to a detailed understanding of how transition metals such as iron, copper, manganese, and zinc are acquired by cells . The regulation of metal transport has been defined at both the transcriptional and posttranslational levels . Results from studies on S . cerevisiae have been used to understand metal transport in other species of yeast as well as in higher eukaryotes.

Protein Sci, 2002 Aug, 11(8), 1971 - 7
Sequence conservation in the chagasin family suggests a common trend in cysteine proteinase binding by unrelated protein inhibitors; Rigden DJ et al.; The recently described inhibitor of cysteine proteinases from Trypanosoma cruzi, chagasin, was found to have close homologs in several eukaryotes, bacteria and archaea, the first protein inhibitors of cysteine proteases in prokaryotes . These previously uncharacterized 110-130 residue-long proteins share a well-conserved sequence motif that corresponds to two adjacent beta-strands and the short loop connecting them . Chagasin-like proteins also have other conserved, mostly aromatic, residues, and share the same predicted secondary structure . These proteins adopt an all-beta fold with eight predicted beta-strands of the immunoglobulin type . The phylogenetic distribution of the chagasins generally correlates with the presence of papain-like cysteine proteases . Previous studies have uncovered similar trends in cysteine proteinase binding by two unrelated inhibitors, stefin and p41, that belong to the cystatin and thyroglobulin families, respectively . A hypothetical model of chagasin-cruzipain interaction suggests that chagasin may dock to the cruzipain active site in a similar manner with the conserved NPTTG motif of chagasin forming a loop that is similar to the wedge structures formed at the active sites of papain and cathepsin L by stefin and p41.

J Bacteriol, 2002 Aug, 184(16), 4594 - 600
Functional annotation of class I lysyl-tRNA synthetase phylogeny indicates a limited role for gene transfer; Ambrogelly A et al.; Functional and comparative genomic studies have previously shown that the essential protein lysyl-tRNA synthetase (LysRS) exists in two unrelated forms . Most prokaryotes and all eukaryotes contain a class II LysRS, whereas most archaea and a few bacteria contain a less common class I LysRS . In bacteria the class I LysRS is only found in the alpha-proteobacteria and a scattering of other groups, including the spirochetes, while the class I protein is by far the most common form of LysRS in archaea . To investigate this unusual distribution we functionally annotated a representative phylogenetic sampling of LysRS proteins . Class I LysRS proteins from a variety of bacteria and archaea were characterized in vitro by their ability to recognize Escherichia coli tRNA(Lys) anticodon mutants . Class I LysRS proteins were found to fall into two distinct groups, those that preferentially recognize the third anticodon nucleotide of tRNA(Lys) (U36) and those that recognize both the second and third positions (U35 and U36) . Strong recognition of U35 and U36 was confined to the pyrococcus-spirochete grouping within the archaeal branch of the class I LysRS phylogenetic tree, while U36 recognition was seen in other archaea and an example from the alpha-proteobacteria . Together with the corresponding phylogenetic relationships, these results suggest that despite its comparative rarity the distribution of class I LysRS conforms to the canonical archaeal-bacterial division . The only exception, suggested from both functional and phylogenetic data, appears to be the horizontal transfer of class I LysRS from a pyrococcal progenitor to a limited number of bacteria.

J Biotechnol, 2002 Jun 13, 96(1), 13 - 21
Prevention and reversion of protein aggregation by molecular chaperones in the E . coli cytosol: implications for their applicability in biotechnology; Schlieker C et al.; The amount of a native protein reflects an equilibrium of protein synthesis, de novo folding and protein stability . Stress situations, like heat shock, or overproduction of a protein can cause an imbalance in this equilibrium, resulting in protein aggregation . Molecular chaperones control protein folding processes and protect misfolded proteins from aggregation in all cells . Since protein aggregation is frequently observed upon synthesis of heterologous proteins in E . coli, molecular chaperones have been applied in biotechnology by their co-overproduction with the desired protein . While increasing protein solubility in some cases, this approach has not been generally successful . Recent findings demonstrate, that protein aggregation, even in case of inclusion bodies, must not be a dead end in the life cycle of a protein . Such resolubilization of aggregated proteins is mediated by a bi-chaperone system consisting of ClpB and DnaK, the prokaryotic representatives of the Hsp100 and Hsp70 families . The disaggregation capacity of this bi-chaperone system has now been demonstrated in vitro and in vivo for a wide variety of aggregated proteins and offers a new perspective to increase the solubility of proteins of interest.

Tsitol Genet, 2002 Mar-Apr, 36(2), 3 - 10
{Screening substances derived from cultures of medicinal plants for antimutagenic activity in the Escherichia coli-bacteriophage lambda system}; Dvornyk AS et al.; In Escherichia coli--bacteriophage lambda system protective properties of the extracts derived from the biomass of cultured Panax ginseng, Polyscias filicifolia, Rhodiola rosea, Ungernia victoris cells, and those from intact Rhodiola roots have been studied . Escherichia coli--bacteriophage lambda system responsiveness was found to vary with the test-object state, namely: the deleted bacteriophage form (lambda-4) as well as previously mutagenized bacteriophage were more sensitive to the mutagenic and antimutagenic influence versus the native bacteriophage lambda + . The contribution of extracts in the induction and realization of the lethal injuries in phages caused by nitrite acid in extracellular phage (conditions in vitro) was estimated thus enabling to discriminate between the protective and antimutagenic extract activities . Protective extract effect in the given test-system appeared to be higher their antimutagenic action . With the most responsive bacteriophage variant the extracts from the biomass of cultured Rh . rosea and P . filicifolia cells showed high protective and somewhat lower antimutagenic activities . With other phages significant antimutagenic potential of extracts was demonstrated, which by their protective effect could be arranged in a raw as follows U . victoris > P . ginseng > P . filicifolia . The primary screening for the antimutagenic effect of preparations in the prokaryotic systems could be reduced to the investigation of their effects on the object inactivation exposed to the mutagen in vitro.

Nucleic Acids Res, 2002 Aug 1, 30(15), 3378 - 86
GenomeHistory: a software tool and its application to fully sequenced genomes; Conant GC et al.; We present a publicly available software tool that identifies all pairs of duplicate genes in a genome and then determines the degree of synonymous and non-synonymous divergence between each duplicate pair . Using this tool, we analyze the relations between (i) gene function and the propensity of a gene to duplicate and (ii) the number of genes in a gene family and the family's rate of sequence evolution . We do so for the complete genomes of four eukaryotes (fission and budding yeast, fruit fly and nematode) and one prokaryote (Escherichia coli) . For some classes of genes we observe a strong relationship between gene function and a gene's propensity to undergo duplication . Most notably, ribosomal genes and transcription factors appear less likely to undergo gene duplication than other genes . In both fission and budding yeast, we see a strong positive correlation between the selective constraint on a gene and the size of the gene family of which this gene is a member . In contrast, a weakly negative such correlation is seen in multicellular eukaryotes.

Genetika, 2002 Jun, 38(6), 793 - 8
{Segmented genome: elementary units of genome structure}; Trifonov EN; Numerous observations, measurements and calculations strongly indicate that both eukaryotic and prokaryotic genomes are built as linear arrays of units of rather uniform size, about 400 base pairs . The units are likely to correspond to early individual genes that existed, presumably, in form of DNA circles . Their combinatorial fusion resulted eventually in formation of the early segmented genomes . The segmented structure of the genomes is, apparently, still maintained by some structural selection pressures . Some of the units can be recognized in the sequences by characteristic sequence motifs at the borders of the units . Identification and characterization of the units, their mapping on the genomes should become an important prerequisite of genome comparisons and genome evolution studies.

Genetika, 2002 Jun, 38(6), 719 - 26
{Acquisition and loss of modules: the construction set of transposable elements}; Capy P et al.; Phylogenetic analysis of transposable elements (TEs) allows us to define the relationships between the domains or gene(s) that compose them . Moreover, modules of a few amino-acids can be detected within gag, pol, env genes or within the integrase domain of retrotransposons and transposase of DNA elements . The combination of these observations clearly shows that the evolutionary history of TEs is the outcome of the acquisition and loss of modules with differing origins and histories . This raises the question of the origin of TEs: are they derived from viruses? Are they where viruses come from? Do the basic building bricks come from the prokaryotes, and can they be assembled in the eukaryotes? Are the TEs found in prokaryotes the result of the disintegration of complex elements such as retroelements? Do they evolve from the simplest to the more complex, or are they opportunistic sequences evolving by acquiring and/or losing modules which may be either important or superfluous to their fitness (i.e., their ability to transpose) . These are some of the questions that are addressed and discussed in the light of the comparative structures of TEs.

J Biol Chem, 2002 Oct 4, 277(40), 37464 - 8 Epub 2002 Jul 23.
A novel ligand-binding domain involved in regulation of amino acid metabolism in prokaryotes; Ettema TJ et al.; A combination of sequence profile searching and structural protein analysis has revealed a novel type of small molecule binding domain that is involved in the allosteric regulation of prokaryotic amino acid metabolism . This domain, designated RAM, has been found to be fused to the DNA-binding domain of Lrp-like transcription regulators and to the catalytic domain of some metabolic enzymes, and has been found as a stand-alone module . Structural analysis of the RAM domain of Lrp reveals a betaalphabetabetaalphabeta-fold that is strikingly similar to that of the recently described ACT domain, a ubiquitous allosteric regulatory domain of many metabolic enzymes . However, structural alignment and re-evaluation of previous mutagenesis data suggest that the effector-binding sites of both modules are significantly different . By assuming that the RAM and ACT domains originated from a common ancestor, these observations suggest that their ligand-binding sites have evolved independently . Both domains appear to play analogous roles in controlling key steps in amino acid metabolism at the level of gene expression as well as enzyme activity.

J Biol Chem, 2002 Sep 27, 277(39), 35847 - 52 Epub 2002 Jul 22.
Binding of ribosome recycling factor to ribosomes, comparison with tRNA; Hirokawa G et al.; The prokaryotic post-termination ribosomal complex is disassembled by ribosome recycling factor (RRF) and elongation factor G . Because of the structural similarity of RRF and tRNA, we compared the biochemical characteristics of RRF binding to ribosomes with that of tRNA . Unesterified tRNA inhibited the disassembly of the post-termination complex in a competitive manner with RRF, suggesting that RRF binds to the A-site . Approximately one molecule of ribosome-bound RRF was detected after isolation of the RRF-ribosome complex . RRF and unesterified tRNA similarly inhibited the binding of N-acetylphenylalanyl-tRNA to the P-site of non-programmed but not programmed ribosomes . Under the conditions in which unesterified tRNA binds to both the P- and E-sites of non-programmed ribosomes, RRF inhibited 50% of the tRNA binding, suggesting that RRF does not bind to the E-site . The results are consistent with the notion that a single RRF binds to the A- and P-sites in a somewhat analogous manner to the A/P-site bound peptidyl tRNA . The binding of RRF and tRNA to ribosomes was influenced by Mg(2+) and NH(4)(+) ions in a similar manner.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1999, 31(3), 264 - 268
Helicobacter pylori HspA Heat-shock Protein Gene Cloning, Expression and Immunogenicity; Li MF et al.; Helicobacter pylori is the causative agent of gastritis and peptic ulcer in human . The heat-shock protein A (HspA)may stimulate the immunoresponse protecting human body against challenge of H.pylori . The gene encoding the structural A subunit of H.pylori heat-shock protein was amplified from H.pylori chromosomal DNA by PCR, and was inserted in the prokaryotic expression vector pET-22b(+), and then was transformed into the BL-21(DE3)E.coli strain to express the HspA recombinant protein . HspA gene was measured to be 354 base pairs, and the recombinant protein gene encoded polypeptides of 118 amino acid residues, corres-ponding to calculated molecular weight of 15 kD . Western blot analysis of HspA recombinant protein was confirmed that it could be specifically recognized by the serum of H.pylori-infected patients, and could also be re-cognized by the serum of immunized Balb/c mice with HspA itself . This result suggests that HspA may be an effective protein vaccine for prevention and treatment of the infection of H.pylori.

Genetics, 2002 Jul, 161(3), 1065 - 75
Formation of large palindromic DNA by homologous recombination of short inverted repeat sequences in Saccharomyces cerevisiae; Butler DK et al.; Large DNA palindromes form sporadically in many eukaryotic and prokaryotic genomes and are often associated with amplified genes . The presence of a short inverted repeat sequence near a DNA double-strand break has been implicated in the formation of large palindromes in a variety of organisms . Previously we have established that in Saccharomyces cerevisiae a linear DNA palindrome is efficiently formed from a single-copy circular plasmid when a DNA double-strand break is introduced next to a short inverted repeat sequence . In this study we address whether the linear palindromes form by an intermolecular reaction (that is, a reaction between two identical fragments in a head-to-head arrangement) or by an unusual intramolecular reaction, as it apparently does in other examples of palindrome formation . Our evidence supports a model in which palindromes are primarily formed by an intermolecular reaction involving homologous recombination of short inverted repeat sequences . We have also extended our investigation into the requirement for DNA double-strand break repair genes in palindrome formation . We have found that a deletion of the RAD52 gene significantly reduces palindrome formation by intermolecular recombination and that deletions of two other genes in the RAD52-epistasis group (RAD51 and MRE11) have little or no effect on palindrome formation . In addition, palindrome formation is dramatically reduced by a deletion of the nucleotide excision repair gene RAD1.

Vet Microbiol, 2002 Aug 25, 88(2), 161 - 73
Experimental transmission of epizootic bovine abortion (foothill abortion); Stott JL et al.; Advances in defining the biology of epizootic bovine abortion (EBA), including identification of the etiologic agent, have been hampered by the inability to reproduce the disease with confidence . Experimental reproduction of EBA, by feeding the tick vector Ornithodoros coriaceus on susceptible pregnant heifers, is not reliable . The primary objectives of this study were to identify specific tissue(s) obtained from EBA-infected fetuses that could transmit the disease, and then utilize such an infectious challenge system to better define the pathogen, host immunity and geographic distribution of the agent . Described here is the ability to routinely reproduce EBA following inoculation of cryopreserved suspensions of homogenized thymus into susceptible pregnant heifers . This challenge system permitted experiments demonstrating the agent was non-filterable, inactivated upon sonication and susceptible to antibiotics . These findings suggest a prokaryotic microbe and represent a major advance in EBA research . Additional experiments demonstrated that inoculation of the cryopreserved EBA-infectious tissue into heifers, prior to breeding, conferred immunity . Furthermore, such immunized heifers were resistant to challenge with heterologous sources of infectious tissue, suggesting monovalent vaccine development might be feasible . Lastly, challenge studies employing animals from Central Nevada, an area considered free of EBA, demonstrated partial immunity, suggesting the pathogen, and possibly the disease, enjoy a broader distribution than previously thought.

Eur J Biochem, 2002 Jul, 269(14), 3409 - 16
Kinetic and biochemical analyses on the reaction mechanism of a bacterial ATP-citrate lyase; Kanao T et al.; The prokaryotic ATP-citrate lyase is considered to be a key enzyme of the carbon dioxide-fixing reductive tricarboxylic acid (RTCA) cycle . Kinetic examination of the ATP-citrate lyase from the green sulfur bacterium Chlorobium limicola (Cl-ACL), an alpha(4)beta(4) heteromeric enzyme, revealed that the enzyme displayed typical Michaelis-Menten kinetics toward ATP with an apparent K(m) value of 0.21 +/- 0.04 mm . However, strong negative cooperativity was observed with respect to citrate binding, with a Hill coefficient (n(H)) of 0.45 . Although the dissociation constant of the first citrate molecule was 0.057 +/- 0.008 mm, binding of the first citrate molecule to the enzyme drastically decreased the affinity of the enzyme for the second molecule by a factor of 23 . ADP was a competitive inhibitor of ATP with a K(i) value of 0.037 +/- 0.006 mm . Together with previous findings that the enzyme catalyzed the reaction only in the direction of citrate cleavage, these kinetic features indicated that Cl-ACL can regulate both the direction and carbon flux of the RTCA cycle in C . limicola . Furthermore, in order to gain insight on the reaction mechanism, we performed biochemical analyses of Cl-ACL . His273 of the alpha subunit was indicated to be the phosphorylated residue in the catalytic center, as both catalytic activity and phosphorylation of the enzyme by ATP were abolished in an H273A mutant enzyme . We found that phosphorylation of the subunit was reversible . Nucleotide preference for activity was in good accordance with the preference for phosphorylation of the enzyme . Although residues interacting with nucleotides in the succinyl-CoA synthetase from Escherichia coli were conserved in AclB, AclA alone could be phoshorylated with the same nucleotide specificity observed in the holoenzyme . However, AclB was necessary for enzyme activity and contributed to enhance phosphorylation and stabilization of AclA.

Biochemistry, 2002 Jul 30, 41(30), 9426 - 30
Evolution of the allosteric ligand sites of mammalian phosphofructo-1-kinase; Kemp RG et al.; Mammalian phosphofructokinase (PFK) has evolved by a process of tandem gene duplication and fusion to yield a protein that is more than double the size of prokaryotic PFKs . On the basis of complete conservation of active site residues in the N-terminal half of the eukaryotic enzyme with those of the bacterial PFKs, one assumes that the active site of the eukaryotic PFK is located in the N-terminal half . Again using sequence comparisons, the four allosteric ligand sites of mammalian PFK have been thought to arise from the duplicated catalytic and regulatory sites of the ancestral PFK . Previous site-directed mutagenesis studies {Li et al . (1999) Biochemistry 38, 16407-16412; Chang and Kemp (2002) Biochem . Biophys . Res . Commun . 290, 670-675} have identified the origins of the citrate and fructose 2,6-bisphosphate sites . Here, site-directed mutagenesis of two arginine residues (Arg-433 and Arg-429) of mouse phosphofructokinase is used to identify the ATP inhibitory site, and, by inference, the AMP/ADP site . Mutation of the residues to alanine reduced ATP inhibition in the case of Arg-429 and eliminated ATP inhibition in the instance of Arg-433 . The Arg-433 mutant could be inhibited by citrate, and that inhibition could be reversed by fructose 2,6-bisphosphate and cyclic AMP, a high-affinity ligand for the AMP/ADP binding site . It is concluded that the two inhibitors, ATP and citrate, of mammalian PFK interact with sites that have evolved from the duplicated phosphoenolpyruvate/ADP allosteric site of the ancestral PFK . The two sites for activators, fructose 2,6-bisphosphate and AMP or ADP, have evolved from the catalytic site of the ancestral precursor.

Front Biosci, 2002 Aug 01, 7, d1815 - 24
Recent progress in Bacillus subtilis two-component regulation; Ogura M et al.; Two-component regulatory systems serve to control gene expression in response to environmental and physiological changes . They are widespread among a variety of organisms and most often found in prokaryotes . One of the gram-positive microorganisms Bacillus subtilis is a well-studied bacterium whose complete nucleotide sequence has been determined . Thus, it is now possible to study transcription of the whole genome with microarray analysis . In this review we summarize the recent progress in B . subtilis two-component regulatory systems by describing the known systems and those for which the function was recently assigned . Also included is an attempt to construct a partial transcriptional network involving several two-component systems . The studies described here are based on the data from traditional genetics and biochemistry, and from microarray analysis of 29 two-component systems.

Proc Natl Acad Sci U S A, 2002 Aug 6, 99(16), 10310 - 5 Epub 2002 Jul 18.
The structure of a replication initiator unites diverse aspects of nucleic acid metabolism; Campos-Olivas R et al.; Rolling circle replication is a mechanism for copying single-stranded genomes by means of double-stranded intermediates . A multifunctional replication initiator protein (Rep) is indispensable for the precise initiation and termination of this process . Despite the ubiquitous presence and fundamental importance of rolling circle replication elements, structural information on their respective replication initiators is still missing . Here we present the solution NMR structure of the catalytic domain of Rep, the initiator protein of tomato yellow leaf curl virus . It is composed of a central five-stranded anti-parallel beta-sheet, flanked by a small two-stranded beta-sheet, a beta-hairpin and two alpha-helices . Surprisingly, the structure reveals that the catalytic Rep domain is related to a large group of proteins that bind RNA or DNA . Identification of Rep as resembling the family of ribonucleoprotein/RNA-recognition motif fold proteins establishes a structure-based evolutionary link between RNA binding proteins, splicing factors, and replication initiators of prokaryotic and eukaryotic single-stranded DNA elements and mammalian DNA tumor viruses.

Biochem Biophys Res Commun, 2002 Jul 26, 295(4), 890 - 7
Expression studies of two paralogous ppa genes encoding distinct Family I pyrophosphatases in marine unicellular cyanobacteria reveal inactivation of the typical cyanobacterial gene; Gomez-Garcia MR et al.; Genome sequence analyses revealed the occurrence of two paralogous ppa genes potentially encoding distinct Family I inorganic pyrophosphatases (sPPases, EC3.6.1.1) in the marine unicellular cyanobacteria Prochlorococcus marinus strains MED4 and MIT9313 and Synechococcus sp . WH8102 . Protein sequence alignment and phylogenetic analysis indicated that the ppa gene proper of cyanobacteria (ppa1) encodes a presumably inactive mutant enzyme whereas the second gene (ppa2) might encode an active sPPase closely related to those of some proteobacteria . Heterologous expression of the two cloned P . marinus MED4 ppa genes in Escherichia coli confirmed this proposal, only the inactive ppa1 product being immunodetected by anti-cyanobacterial sPPase antibodies . A possible scenario of ppa gene inactivation and replacement in the context of the postulated rapid diversification of marine unicellular cyanobacteria, the most abundant photosynthetic prokaryotes in the oceans, is discussed.

FEMS Microbiol Lett, 2002 Jul 16, 213(1), 121 - 6
Nitric oxide releases intracellular zinc from prokaryotic metallothionein in Escherichia coli; Binet MR et al.; Nitric oxide (NO) has a broad spectrum of signalling and regulatory functions and multiple molecular targets . Recently, the intrabacterial toxicity of NO and mechanisms for NO resistance have been intensively investigated . Here we report for the first time that NO elicits release of zinc from a bacterial protein . Using the zinc-responsive expression of zntA (encoding a Zn-exporting P-type ATPase) fused to lacZ, i.e . Phi(zntA-lacZ), to monitor intracellular zinc, and SmtA (the Synechococcus metallothionein) as zinc store, we have shown that the NO donors NOC-5 and NOC-7 elicit zinc ejection . No increase in Phi(zntA-lacZ) activity was observed in a zntR mutant, indicating the specificity of the zntA promoter response to zinc ions.

J Org Chem, 2002 Jul 26, 67(15), 5352 - 8
Synthesis of the tRNA(Lys,3) anticodon stem-loop domain containing the hypermodified ms2t6A nucleoside; Bajji AC et al.; The synthesis of a protected form of the hypermodified nucleoside, N-{(9-beta-D-ribofuranosyl-2-methylthiopurin-6-yl)carbamoyl}threonine, (ms2t6A) is reported . The hypermodified nucleoside was subsequently elaborated to the protected nucleoside phosphophoramidite using a protecting group strategy compatible with standard RNA oligonucleotide chemistry . The phosphoramidite reagent was then used to synthesize the 17-nucleotide RNA hairpin having the sequence of the anticodon stem-loop (ASL) domain of human tRNA(Lys,3), the primer for HIV-1 reverse transcriptase . Introduction of the modification at position 37 of the tRNA ASL modestly decreases the thermodynamic stability of the RNA hairpin as has been seen previously for the prokaryotic t6A nucleoside lacking the 2-methylthio substituent . 2D NOESY NMR spectra of the ms2t6A containing tRNA ASL indicate that the threonyl side chain adopts a conformation similar to that seen in the solution structure of the analogous t6A containing E . coli tRNA(Lys), despite the presence of the bulky methylthio group . This synthetic approach allows for site-specific incorporation of the hypermodified nucleoside and should facilitate future studies directed at understanding the roles of nucleoside modification in modulating the stability and specificity of biologically important RNA-RNA interactions . Our synthesis of the ms2t6A containing RNAs demonstrates that this methodology is suitable for obtaining quantities of RNA required for structural studies of the HIV primer tRNA.

Mol Membr Biol, 2002 Apr-Jun, 19(2), 137 - 47
Voltage-gated H+ channels associated with human phagocyte superoxide-generating NADPH oxidases: sequence comparisons, structural predictions, and phylogenetic analyses; Kimball RA et al.; The N-terminal domain of the human phagocyte flavocytochrome b558 NADPH oxidase, gp91phox, is believed to be a heme-containing voltage-gated H+ channel . The authors have conducted structural, sequence and phylogenetic analyses of the putative transmembrane channel/heme-binding domains of all homologous proteins in the NCBI GenBank database as of May 2001, as well as of the full-length proteins . Fifty-six homologues were identified, including 26 from animals, 19 from plants, seven from yeast, one from a slime mould and three from bacteria . Six well-defined sub-families were revealed by phylogenetic tree construction, two consisting of animal proteins, two of plant proteins, and one each of yeast and bacterial homologues, with the slime mould protein clustering loosely with one of the animal clusters . Signature sequences for the entire family as well as for the sub-families were determined . Most proteins have six putative TMSs, four of which may comprise the heme-binding H+ channel . The hydrophobic and amphipathic characteristics of each of the putative alpha-helical transmembrane segments were defined, and conserved residues that may be involved in heme binding, channel formation, and/or conformational changes were identified . The analyses lead to the suggestion that the oxidase domain became associated with the channel/heme-binding domain to form a single polypeptide chain early in evolutionary history, before eukaryotes diverged from prokaryotes, and that genetic transmission to present day organisms occurred primarily by vertical descent.

Environ Microbiol, 2002 Jul, 4(7), 422 - 9
Development of a novel, bioluminescence-based, fungal bioassay for toxicity testing; Weitz HJ et al.; Naturally bioluminescent fungi, Armillaria mellea and Mycena citricolor, were used to develop a novel, bioluminescence-based bioassay for toxicity testing . Bioassays were carried out to assess the toxicity of 3,5-dichlorophenol (3,5-DCP), pentachlorophenol (PCP), copper and zinc . The results suggested that 60 min was a suitable exposure time for the bioassay . Light reduction was observed in response to 3,5-DCP, PCP and Cu for both A . mellea and M . citricolor, but to Zn only for A . mellea . Armillaria mellea was significantly less sensitive to 3,5-DCP and PCP than M . citricolor . The EC50 values for A . mellea and M . citricolor were similar to EC50 values for 3,5-DCP, PCP and Cu (but not Zn) of bioluminescence-based bacterial biosensors . They were also similar to EC50 values for Cu and Zn of a bioluminescence-based yeast biosensor . The results highlighted the importance of using both prokaryotic and eukaryotic biosensors . The novel bioassay provides a rapid and sensitive method to assess bioavailability of pollutants as well as a method to determine their toxicity to filamentous fungi . It also expands the range of organisms that can be used for bioluminescence-based toxicity testing by complementing existing biosensors.

J Biol Chem, 2002 Sep 20, 277(38), 35248 - 56 Epub 2002 Jul 16.
A cold-regulated nucleic acid-binding protein of winter wheat shares a domain with bacterial cold shock proteins; Karlson D et al.; The molecular mechanisms of cold acclimation are still largely unknown; however, it has been established that overwintering plants such as winter wheat increases freeze tolerance during cold treatments . In prokaryotes, cold shock proteins are induced by temperature downshifts and have been proposed to function as RNA chaperones . A wheat cDNA encoding a putative nucleic acid-binding protein, WCSP1, was isolated and found to be homologous to the predominant CspA of Escherichia coli . The putative WCSP1 protein contains a three-domain structure consisting of an N-terminal cold shock domain with two internal conserved consensus RNA binding domains and an internal glycine-rich region, which is interspersed with three C-terminal CX(2)CX(4)HX(4)C (CCHC) zinc fingers . Each domain has been described independently within several nucleotide-binding proteins . Northern and Western blot analyses showed that WCSP1 mRNA and protein levels steadily increased during cold acclimation, respectively . WCSP1 induction was cold-specific because neither abscisic acid treatment, drought, salinity, nor heat stress induced WCSP1 expression . Nucleotide binding assays determined that WCSP1 binds ssDNA, dsDNA, and RNA homopolymers . The capacity to bind dsDNA was nearly eliminated in a mutant protein lacking C-terminal zinc fingers . Structural and expression similarities to E . coli CspA suggest that WCSP1 may be involved in gene regulation during cold acclimation.

Antimicrob Agents Chemother, 2002 Aug, 46(8), 2490 - 7
Stress-based identification and classification of antibacterial agents: second-generation Escherichia coli reporter strains and optimization of detection; Shapiro E et al.; Escherichia coli strains bearing single-copy fusions between the lacZ reporter gene and the cspA, ibp, or P3rpoH stress promoters offer a simple means to detect sublethal concentrations of antibacterial agents interfering with prokaryotic translation or cell envelope integrity while simultaneously providing information on the mechanism of action of the test compound (A . A . Bianchi and F . Baneyx, Appl . Environ . Microbiol . 65:5023-5027, 1999) . Here, we expand the usefulness of this system by (i) demonstrating that a fusion between the SOS-inducible sulA promoter and lacZ is a highly specific probe for the detection of antimicrobial agents that ultimately interfere with DNA replication, (ii) showing that inactivation of the tolC gene allows efficient detection of very low concentrations of model antibiotics (including aminoglycosides) whereas polymyxin B-mediated outer membrane permeabilization facilitates the identification of intermediate concentrations of hydrophobic compounds, and (iii) validating the potential of detector strains and sensitization strategies for high-throughput screening using a reproducible and internally consistent 96-well microplate assay.

J Inorg Biochem, 2002 Jul 25, 91(1), 78 - 86
Engineering the proximal heme cavity of catalase-peroxidase; Jakopitsch C et al.; Catalase-peroxidases (KatGs) are prokaryotic heme peroxidases with homology to yeast cytochrome c peroxidase (CCP) and plant ascorbate peroxidases (APXs) . KatGs, CCP and APXs contain identical amino acid triads in the heme pocket (distal Arg/Trp/His and proximal His/Trp/Asp), but differ dramatically in their reactivities towards hydrogen peroxide and various one-electron donors . Only KatGs have high catalase activity in addition to a peroxidase activity of broad specificity . Here, we investigated the effect of mutating the conserved proximal triad on KatG catalysis . With the exception of W341F, all variants (H290Q, W341A, D402N, D402E) exhibited a catalase activity <1% of wild-type KatG and spectral properties indicating alterations in heme coordination and spin states . Generally, the peroxidase activity was much less effected by these mutations . Compared with wild-type KatG the W341F variant had a catalase and halogenation activity of about 40% and an even increased overall peroxidase activity . This variant, for the first time, allowed to monitor the hydrogen peroxide mediated transitions of ferric KatG to compound I and back to the resting enzyme . Compound I reduction by aromatic one-electron donors (o-dianisidine, pyrogallol, aniline) was not influenced by exchanging Trp by Phe . The findings are discussed in comparison with the data known from CCP and APX and a reaction mechanism for the multifunctional activity of the W341F variant is suggested.

J Biol Chem, 2002 Sep 27, 277(39), 36760 - 5 Epub 2002 Jul 15.
Receptor methylation controls the magnitude of stimulus-response coupling in bacterial chemotaxis; Levit MN et al.; Motile prokaryotes employ a chemoreceptor-kinase array to sense changes in the media and properly adjust their swimming behavior . This array is composed of a family of Type I membrane receptors, a histidine protein kinase (CheA), and an Src homology 3-like protein (CheW) . Binding of an attractant to the chemoreceptors inhibits CheA, which results in decreased phosphorylation of the chemotaxis response regulator (CheY) . Sensitivity of the system to stimuli is modulated by a protein methyltransferase (CheR) and a protein methylesterase (CheB) that catalyze the methylation and demethylation of specific glutamyl residues in the cytoplasmic domain of the receptors . One of the most fundamental unanswered questions concerning the bacterial chemotaxis mechanism is the quantitative relationship between ligand binding to receptors and CheA inhibition . We show that the receptor glutamyl modifications cause adaptation by changing the gain (magnitude amplification) between attractant binding and kinase inhibition without substantially affecting ligand binding affinity . The mechanism adjusts receptor sensitivity to background stimulus intensity over several orders of magnitude of attractant concentrations . The cooperative effects of ligand binding appear to be minimal with Hill coefficients for kinase inhibition less than 2, independent of the state of glutamyl modification.

Cell Res, 2002 Jun, 12(2), 157 - 60
Get effective polyclonal antisera in one month; Hu YX et al.; According to the traditional immunization procedure, after the first injection of the sample A (emulsion of aimed antigen and Freund's complete adjuvant) to immunize rabbit, successive injections of the sample B (emulsion of aimed antigen and Freund's incomplete adjuvant) were followed every 2-4 weeks . In general, high titer of the corresponding polyclonal antisera will be observed after 4-5 injections of sample B in 3-4 months . This report presents a simply modified procedure that was able to stimulate the antisera formation in one month and achieve enough avidity to satisfy either Western blot or immunohistochemistry analysis . It just applied an additional injection of the sample A to the rabbit at the 3rd day after the primary immunization injection . You could gain the high titer of the antisera right after the first sample B injection in one month . This method has produced the desired results in three different recombinant antigens with different molecular weight (5.9 KD-55 KD) expressed from prokaryotic or eukaryotic cells.

Infect Immun, 2002 Aug, 70(8), 4124 - 31
Disruption of the gene homologous to mammalian Nramp1 in Mycobacterium tuberculosis does not affect virulence in mice; Boechat N et al.; Natural-resistance-associated macrophage protein 1 (Nramp1) is a divalent cation transporter belonging to a family of transporter proteins highly conserved in eukaryotes and prokaryotes . Mammalian and bacterial transporters may compete for essential metal ions during mycobacterial infections . The mycobacterial Nramp homolog may therefore be involved in Mycobacterium tuberculosis virulence . Here, we investigated this possibility by inactivating the M . tuberculosis Nramp1 gene (Mramp) by allelic exchange mutagenesis . Disruption of Mramp did not affect the extracellular growth of bacteria under standard conditions . However, the Mramp mutation was associated with growth impairment under conditions of limited iron availability . The Mramp mutant displayed no impairment of growth or survival in macrophages derived from mouse bone marrow or in Nramp1(+/+) and Nramp1(-/-) congenic murine macrophage cell lines . Following intravenous challenge in BALB/c mice, counts of parental and Mramp mutant strains were similar in the lungs and spleens of the animals at all time points studied . These results indicate that Mramp does not contribute to the virulence of M . tuberculosis in mice.

Syst Biol, 2001 Aug, 50(4), 497 - 512
Genomic and phylogenetic perspectives on the evolution of prokaryotes; Brown JR; Prokaryotes have been at the forefront of the genome sequencing revolution . Many genomes have been completely sequenced, revealing much about bacterial and archaeal genome content and organization . Yet, a meaningful evolutionary picture of prokaryotes still eludes us . Much of the problem lies in understanding the mode and tempo of genome evolution . Here phenylalanyl-tRNA synthetase is used as an example of the complex interplay among lateral gene transfer, operon recombination, and gene recruitment in the evolution of some prokaryotic genes . Promising new approaches to genomic analyses, which could add to our understanding prokaryotic evolution and help in their classification, are discussed.

Perspect Biol Med, 2002 Summer, 45(3), 359 - 76
Thomas Kuhn is alive and well: the evolutionary relationships of simple life form--a paradigm under siege ?
Lyons SL.
A Kuhnian framework is used to analyze the current controversy over whether two or three fundamental types of life forms exist . Until the 1980s, all life was classified into two primary forms: eukaryotes and prokaryotes . Using molecular sequencing data, Carl Woese suggested the archaebacteria constituted a third domain . In the mid-1990s, Radhey S . Gupta challenged the three-domain hypothesis . While this dispute may seem to be a purely technical debate over the analysis of protein and nucleic acid sequence data, the controversy encompasses broader issues such as the aims of classification and the role of microorganisms in the biosphere . At the heart of this dispute is what kinds of data are relevant to constructing an overall taxonomy . The prestige of molecular biology played a large role in why the three-domain hypothesis was accepted so readily, but supporters of the two-domain hypothesis argue that the fossil record, morphology, and cell physiology should all play a role in taxonomy . This case study provides a good example of a paradigm shift in the making, demonstrating that issues beyond the raw data will be significant factors in deciding whether the three-domain hypothesis will prevail or a new classificatory scheme will emerge.

Dis Aquat Organ, 2002 Jun 3, 49(3), 207 - 19
Ultrastructure of a haplosporidian containing Rickettsiae, associated with mortalities among cultured paua Haliotis iris; Hine PM et al.; Uninucleate and multinucleate stages of a protozoan parasite are described from cultured abalone Haliotis iris Martyn, 1784 in New Zealand . The parasite is identified as a haplosporidian by the occurrence of multinucleate plasmodia, mitochondria with tubular cristae, lipid droplets, anastomosing endoplasmic reticulum (aER), multivesicular bodies (MVBs), haplosporogenesis by the production of haplosporosome-like bodies from nuclear membrane-bound Golgi, and their maturation to haplosporosomes . Coated pits occurred in the plasma membrane and coated vesicles were scattered in the cytoplasm, particularly in association with the Golgi face away from the nucleus, and aER . It is concluded that the outward face of the Golgi may be the trans face, and that aER is the trans-Golgi network . Coated pits and bristle-coated vesicles are reported from a haplosporidian for the first time . The vesicles in the MVBs resembled the cores and inner membranes of haplosporosomes, without the outer layer . The possible inter-relationships of these features are discussed . The abalone parasite differs from previously described haplosporidians in the apparent absence of a persistent mitotic spindle, and the presence of intracytoplasmic coccoid to rod-shaped bacteria resembling Rickettsiales-like prokaryotes . Phylogenetic analysis of the 16S rRNA gene sequence of the Rickettsiales-like prokaryotes indicated that these organisms belong to the Rickettsia cluster . The prokaryotes have a high (7%) sequence divergence from known Rickettsieae, with Rickettsia sp . and R . massiliae being the closest relatives . The lack of non-molecular evidence prevents us from proposing a new rickettsial genus at this time.

Proteomics, 2002 Jun, 2(6), 611 - 23
The post-genomic era of interactive proteomics: facts and perspectives; Auerbach D et al.; The availability of completed genome sequences of several eukaryotic and prokaryotic species has shifted the focus towards the identification and characterization of all gene products that are expressed in a given organism . In order to cope with the huge amounts of data that have been provided by large-scale sequencing projects, high-throughout methodologies also need to be applied in the emerging field of proteomics . In this review, we discuss methods that have been recently developed in order to characterize protein interactions and their functional relevance on a large scale . We then focus on those methodologies that are suitable for the identification and characterization of protein-protein interactions, namely the yeast two-hybrid system and related methods . Several recent studies have demonstrated the power of automated approaches involving the yeast two-hybrid system in building so-called "interaction networks", which hold the promise of identifying the entirety of all interactions that take place between proteins expressed in a certain cell type or organism . We compare the yeast two-hybrid system with several other screening methods that have been developed to investigate interactions between proteins that are not amenable to conventional yeast two-hybrid screenings, such as transcriptional activators and integral membrane proteins . The eventual adaptation of such methods to a high-throughput format and their use in combination with automated yeast two-hybrid screenings will help in elucidating protein-protein interactions on a scale that would have been unthinkable just a few years ago.

Bioessays, 2002 Jun, 24(6), 512 - 8
Memory in bacteria and phage; Casadesus J et al.; Whenever the state of a biological system is not determined solely by present conditions but depends on its past history, we can say that the system has memory . Bacteria and bacteriophage use a variety of memory mechanisms, some of which seem to convey adaptive value . A genetic type of heritable memory is the programmed inversion of specific DNA sequences, which causes switching between alternative patterns of gene expression . Heritable memory can also be based on epigenetic circuits, in which a system with two possible steady states is locked in one or the other state by a positive feedback loop . Epigenetic states have been observed in a variety of cellular processes, and are maintained by diverse mechanisms . Some of these involve alternative DNA methylation patterns that are stably transmitted to daughter molecules and can affect DNA-protein interactions (e.g., gene transcription) . Other mechanisms exploit autocatalytic loops whereby proteins establish the proper conditions for their continued synthesis . Template polymers other than nucleic acids (e.g., components of the cell wall) may also propagate epigenetic states . Non-heritable memory is exemplified by parasitic organisms that bear a signature of their previous host, such as host-controlled modification of phage DNA or porin hitchhiking in predatory bacteria . The heterogeneous nature of the examples known may be indicative of widespread occurrence of memory mechanisms in bacteria and phage . However, the actual extent, variety and potential selective value of prokaryotic memory devices remain open questions, still to be addressed experimentally .

J Biol Chem, 2002 Sep 20, 277(38), 34896 - 901 Epub 2002 Jul 10.
Cloning, characterization, and functional studies of a 47-kDa platelet receptor for type III collagen; Chiang TM et al.; A 1.2-kb cDNA fragment encoding a platelet 47-kDa protein has been isolated from a human bone marrow cDNA library by using a degenerate oligonucleotide of the sequenced amino terminus of the purified platelet protein with a poly(dT)(12).(dG) by polymerase chain reaction . A computer search revealed that the cDNA represents the coding sequence of a protein with a fragmentary homology to several proteins . Using a prokaryotic expression system, pBad TOPO-47 cDNA, a 47-kDa recombinant protein was obtained and purified to apparent homogeneity by nickel-nitrilotriacetic acid resin and collagen affinity column . The recombinant protein binds to type III but not type I collagen-Sepharose 2B affinity columns . Anti-47-kDa but not anti-65-kDa antibody inhibits the binding of the recombinant protein to the type III collagen-coated micro titer wells in a dose-dependent manner . Like the receptor protein purified from platelet membranes, the recombinant protein inhibits type III collagen-induced platelet aggregation also in a dose-dependent manner . We have defined two active peptides from the cloned deduced amino acid sequence . Both peptides inhibit type III but not type I collagen-induced platelet aggregation in a dose-dependent fashion . These results suggest that the active binding site of the platelet receptor to type III collagen resides in these portions of the protein.

EMBO J, 2002 Jul 15, 21(14), 3841 - 51
tadA, an essential tRNA-specific adenosine deaminase from Escherichia coli; Wolf J et al.; We report the characterization of tadA, the first prokaryotic RNA editing enzyme to be identified . Escherichia coli tadA displays sequence similarity to the yeast tRNA deaminase subunit Tad2p . Recombinant tadA protein forms homodimers and is sufficient for site-specific inosine formation at the wobble position (position 34) of tRNA(Arg2), the only tRNA having this modification in prokaryotes . With the exception of yeast tRNA(Arg), no other eukaryotic tRNA substrates were found to be modified by tadA . How ever, an artificial yeast tRNA(Asp), which carries the anticodon loop of yeast tRNA(Arg), is bound and modified by tadA . Moreover, a tRNA(Arg2) minisubstrate containing the anticodon stem and loop is sufficient for specific deamination by tadA . We show that nucleotides at positions 33-36 are sufficient for inosine formation in mutant Arg2 minisubstrates . The anticodon is thus a major determinant for tadA substrate specificity . Finally, we show that tadA is an essential gene in E.coli, underscoring the critical function of inosine at the wobble position in prokaryotes.

J Biol Chem, 2002 Aug 30, 277(35), 32400 - 4 Epub 2002 Jun 24.
ATP stimulates signal recognition particle (SRP)/FtsY-supported protein integration in chloroplasts; Yuan J et al.; The signal recognition particle (SRP) and its receptor (FtsY in prokaryotes) are essential for cotranslational protein targeting to the endoplasmic reticulum in eukaryotes and the cytoplasmic membrane in prokaryotes . An SRP/FtsY-like protein targeting/integration pathway in chloroplasts mediates the posttranslational integration of the light-harvesting chlorophyll a/b-binding protein (LHCP) into thylakoid membranes . GTP, chloroplast SRP (cpSRP), and chloroplast FtsY (cpFtsY) are required for LHCP integration into thylakoid membranes . Here, we report the reconstitution of the LHCP integration reaction with purified recombinant proteins and salt-washed thylakoids . Our data demonstrate that cpSRP and cpFtsY are the only soluble protein components required for LHCP integration . In addition, our studies reveal that ATP, though not absolutely required, remarkably stimulates LHCP integration into salt-washed thylakoids . ATP stimulates LHCP integration by a mechanism independent of the thylakoidal pH gradient (DeltapH) and exerts no detectable effect on the formation of the soluble LHCP-cpSRP-targeting complex . Taken together, our results indicate the participation of a thylakoid ATP-binding protein in LHCP integration.

Chem Immunol, 2002, 81, 167 - 206
Toxins of filamentous fungi; Bhatnagar D et al.; Mycotoxins are low-molecular-weight secondary metabolites of fungi . The most significant mycotoxins are contaminants of agricultural commodities, foods and feeds . Fungi that produce these toxins do so both prior to harvest and during storage . Although contamination of commodities by toxigenic fungi occurs frequently in areas with a hot and humid climate (i.e . conditions favorable for fungal growth), they can also be found in temperate conditions . Production of mycotoxins is dependent upon the type of producing fungus and environmental conditions such as the substrate, water activity (moisture and relative humidity), duration of exposure to stress conditions and microbial, insect or other animal interactions . Although outbreaks of mycotoxicoses in humans have been documented, several of these have not been well characterized, neither has a direct correlation between the mycotoxin and resulting toxic effect been well established in vivo . Even though the specific modes of action of most of the toxins are not well established, acute and chronic effects in prokaryotic and eukaryotic systems, including humans have been reported . The toxicity of the mycotoxins varies considerably with the toxin, the animal species exposed to it, and the extent of exposure, age and nutritional status . Most of the toxic effects of mycotoxins are limited to specific organs, but several mycotoxins affect many organs . Induction of cancer by some mycotoxins is a major concern as a chronic effect of these toxins . It is nearly impossible to eliminate mycotoxins from the foods and feed in spite of the regulatory efforts at the national and international levels to remove the contaminated commodities . This is because mycotoxins are highly stable compounds, the producing fungi are ubiquitous, and food contamination can occur both before and after harvest . Nevertheless, good farm management practices and adequate storage facilities minimize the toxin contamination problems . Current research is designed to develop natural biocontrol competitive fungi and to enhance host resistance against fungal growth or toxin production . These efforts could prevent toxin formation entirely . Rigorous programs for reducing the risk of human and animal exposure to contaminated foods and feed also include economically feasible and safe detoxification processes and dietary modifications . Although risk assessment has been made for some mycotoxins, additional, systematic epidemological data for human exposure is needed for establishing toxicological parameters for mycotoxins and the safe dose for humans . It is unreasonable to expect complete elimination of the mycotoxin problem . But multiple approaches will be needed to minimize the economic impact of the toxins on the entire agriculture industry and their harmfulness to human and animal health.

Microbiology, 2002 Jul, 148(Pt 7), 2135 - 47
Biosynthesis of sulfur-containing amino acids in Streptomyces venezuelae ISP5230: roles for cystathionine beta-synthase and transsulfuration; Chang Z et al.; A 0.5 kb fragment of Streptomyces venezuelae ISP5230 genomic DNA was amplified by PCR using primers based on consensus sequences of cysteine synthase isozyme A from bacteria . The deduced amino acid sequence of the PCR product resembled not only cysteine synthase sequences from prokaryotes and eukaryotes but also eukaryotic cystathionine beta-synthase sequences . Probing an Str . venezuelae genomic library with the PCR product located a hybridizing colony from which pJV207 was isolated . Sequencing and analysis of the Str . venezuelae DNA insert in pJV207 detected two ORFs . The deduced amino acid sequence of ORF1 matched both cysteine synthase and cystathionine beta-synthase sequences in GenBank, but its size favoured assignment as a cystathionine beta-synthase . ORF2 in the pJV207 insert was unrelated in function to ORF1; in its sequence the deduced product resembled acetyl-CoA transferases, but disruption of the ORF did not cause a detectable phenotypic change . Disruption of ORF1 failed to elicit cysteine auxotrophy in wild-type Str . venezuelae, but in the cys-28 auxotroph VS263 it prevented restoration of prototrophy with homocysteine or methionine supplements . The change in phenotype implicated loss of the transsulfuration activity that in the wild-type converts these supplements to cysteine . This study concludes that disruption of ORF1 inactivates a cbs gene, the product of which participates in cysteine synthesis by transsulfuration . Enzyme assays of Str . venezuelae mycelial extracts confirmed the formation of cysteine by thiolation of O-acetylserine, providing the first unambiguous detection of this activity in a streptomycete . Enzyme assays also detected cystathionine gamma-synthase, cystathionine beta-lyase and cystathionine gamma-lyase activity in the extracts and showed that the substrate for cystathionine gamma-synthase was O-succinyl-homoserine . Based on assay results, the cys-28 mutation in Str . venezuelae VS263 does not inactivate the cysteine synthase gene but impairs expression in cultures grown in minimal medium.

Mol Microbiol, 2002 Jul, 45(1), 145 - 53
An antiport mechanism for a member of the cation diffusion facilitator family: divalent cations efflux in exchange for K+ and H+; Guffanti AA et al.; Members of the cation diffusion facilitator (CDF) family of membrane transport proteins are found in eukaryotes and prokaryotes . The family encompasses transporters of zinc ions, with cobalt, cadmium and lead ions being additional substrates for some prokaryotic examples . No transport mechanism has previously been established for any CDF protein . It is shown here that the CzcD protein of Bacillus subtilis, a CDF protein, uses an antiporter mechanism, catalysing active efflux of Zn2+ in exchange for K+ and H+ . The exchange is probably electroneutral, energized by the transmembrane pH gradient and oppositely oriented gradients of the other cation substrates . The data suggest that Co2+ and Cd2+ are additional cytoplasmic substrates for CzcD . A second product of the same operon that encodes czcD has sequence similarity to oxidoreductases and is here designated CzcO . CzcO modestly enhances the activity of CzcD but is not predicted to be an integral membrane protein and has no antiport activity of its own.

Mol Microbiol, 2002 Jul, 45(1), 59 - 71
Discovery of two novel families of proteins that are proposed to interact with prokaryotic SMC proteins, and characterization of the Bacillus subtilis family members ScpA and ScpB; Soppa J et al.; Structural maintenance of chromosomes (SMC) proteins are present in all eukaryotes and in many prokaryotes . Eukaryotic SMC proteins form complexes with various non-SMC subunits, which affect their function, whereas the prokaryotic homologues had no known non-SMC partners and were thought to act as simple homodimers . Here we describe two novel families of proteins, widespread in archaea and (Gram-positive) bacteria, which we denote 'segregation and condensation proteins' (Scps) . ScpA genes are localized next to smc genes in nearly all SMC- containing archaea, suggesting that they belong to the same operon and are thus involved in a common process in the cell . The function of ScpA was studied in Bacillus subtilis, which also harbours a well characterized smc gene . Here we show that scpA mutants display characteristic phenotypes nearly identical to those of smc mutants, including temperature- sensitive growth, production of anucleate cells, formation of aberrant nucleoids, and chromosome splitting by the so-called guillotine effect . Thus, both SMC and ScpA are required for chromosome segregation and condensation . Interestingly, mutants of another B . subtilis gene, scpB, which is localized downstream from scpA, display the same phenotypes, which indicate that ScpB is also involved in these functions . ScpB is generally present in species that also encode ScpA . The physical interaction of ScpA and SMC was proven (i) by the use of the yeast two-hybrid system and (ii) by the isolation of a complex containing both proteins from cell extracts of B . subtilis . By extension, we speculate that interaction of orthologues of the two proteins is important for chromosome segregation in many archaea and bacteria, and propose that SMC proteins generally have non-SMC protein partners that affect their function not only in eukaryotes but also in prokaryotes.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2000, 32(2), 126 - 132
Biochemical Characterization of PAI-2 and Its Mutants; Tian Y et al.; To study and compare the biochemical characterization of PAI-2 and its mutants, PCR and site-directed mutagenesis methods were used to generate two PAI-2 mutants, PAI-2CD and PAI-2Q, respectively . The two mutant cDNAs were inserted into prokaryotic expression vector and expressed in a special strain of E.coli, JF1125 . The expected PAI-2 mutant proteins were identified by SDS-PAGE, both covering about 14% of total bacteria proteins . The antigenicity and activity inhibiting uPA of the two mutant proteins were verified to be identical with that of wild PAI-2 by using Western blot and milk-agarose plate assay and reverse milk-agarose zymograph . The harvested recombinant bacterial cells growing in 5 L fermentor were homogenized and purified by the protocols including ammonium sulfate precipitation, Sephadex G-75 gel filtration, Q-Sepharose ion-exchange chromatography and hydrophobic interaction chromatography . The purity of the purified PAI-2CD and PAI-2Q was up to 98% and 90%, the protein yield was 18.4% and 22.1%, the specific activity was 28 640 u/mg and 14 836 u/mg, respectively . The results indicate that the biochemical characterization of PAI-2 mutants was very similar to those of the wide-type PAI-2 except that PAI-2Q can not be catalyzed by tTG.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2002 Jul, 34(4), 498 - 501
Expression, purification and activity measurement of soluble BACE protein in E.coli; Wei XC et al.; To express BACE (beta-site APP cleaving enzyme), an aspartic protease related to Alzheimer's disease in E.coli to obtain the active protein after refolding and purification, the sequence for soluble form of BACE was inserted into prokaryotic expression vector pET11a . After expression in E.coli BL21(DE3), the protein was obtained as inclusion bodies, after refolding and purification . The proteolytic activity of the protein was measured by HPLC and MS . After refolding and purification, the protein exhibited beta-secretase activity by cleaving the synthetic peptide, which was designed according to the beta-secretase site at Swedish mutant of APP, and according Hanes method the kinetics constants for the synthetic peptide of BACE protein were measured . The study will facilitate BACE protein structure determination and inhibitor development efforts, which may lead to the evolution of promising and effective treatments for Alzheimer's disease.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2002 Jul, 34(4), 395 - 9
The progress in mechanism of selenoprotein biosynthesis; Jiang ZH et al.; As the twenty-first amino acid, selenocysteine can be co-translationally incorporated into the polypeptide chain at UGA codon in the coding region of selenoprotein mRNA . The incorporation of selenocysteine needs a cis-acting element SECIS and four gene products: SelA, SelB, SelC and SelD . The position of SECIS in the mRNA of prokaryote and its structural features are greatly different from that of eukaryote . The researchers have made exploration in selenoprotein engineering by virtue of the mechanism of selenocysteine incorporation in Escherichia coli.

J Biol Chem, 2002 Sep 20, 277(38), 35541 - 9 Epub 2002 Jul 02.
Identification of mammalian mitochondrial translational initiation factor 3 and examination of its role in initiation complex formation with natural mRNAs; Koc EC et al.; Human mitochondrial translational initiation factor 3 (IF3(mt)) has been identified from the human expressed sequence tag data base . Using consensus sequences derived from conserved regions of the bacterial IF3, several partially sequenced cDNA clones were identified, and the complete sequence was assembled in silico from overlapping clones . IF3(mt) is 278 amino acid residues in length . MitoProt II predicts a 97% probability that this protein will be localized in mitochondria and further predicts that the mature protein will be 247 residues in length . The cDNA for the predicted mature form of IF3(mt) was cloned, and the protein was expressed in Escherichia coli in a His-tagged form . The mature form of IF3(mt) has short extensions on the N and C termini surrounding a region homologous to bacterial IF3 . The region of IF3(mt) homologous to prokaryotic factors ranges between 21-26% identical to the bacterial proteins . Purified IF3(mt) promotes initiation complex formation on mitochondrial 55 S ribosomes in the presence of mitochondrial initiation factor 2 (IF2(mt)), {(35)S}fMet-tRNA, and either poly(A,U,G) or an in vitro transcript of the cytochrome oxidase subunit II gene as mRNA . IF3(mt) shifts the equilibrium between the 55 S mitochondrial ribosome and its subunits toward subunit dissociation . In addition, the ability of E . coli initiation factor 1 to stimulate initiation complex formation on E . coli 70 S and mitochondrial 55 S ribosomes was investigated in the presence of IF2(mt) and IF3(mt).

Comp Biochem Physiol A Mol Integr Physiol, 2002 Aug, 132(4), 739 - 61
Climate variations and the physiological basis of temperature dependent biogeography: systemic to molecular hierarchy of thermal tolerance in animals; Portner HO; The physiological mechanisms limiting and adjusting cold and heat tolerance have regained interest in the light of global warming and associated shifts in the geographical distribution of ectothermic animals . Recent comparative studies, largely carried out on marine ectotherms, indicate that the processes and limits of thermal tolerance are linked with the adjustment of aerobic scope and capacity of the whole animal as a crucial step in thermal adaptation on top of parallel adjustments at the molecular or membrane level . In accordance with Shelford's law of tolerance decreasing whole animal aerobic scope characterises the onset of thermal limitation at low and high pejus thresholds (pejus=getting worse) . The drop in aerobic scope of an animal indicated by falling oxygen levels in the body fluids and or the progressively limited capacity of circulatory and ventilatory mechanisms . At high temperatures, excessive oxygen demand causes insufficient oxygen levels in the body fluids, whereas at low temperatures the aerobic capacity of mitochondria may become limiting for ventilation and circulation . Further cooling or warming beyond these limits leads to low or high critical threshold temperatures (T(c)) where aerobic scope disappears and transition to an anaerobic mode of mitochondrial metabolism and progressive insufficiency of cellular energy levels occurs . The adjustments of mitochondrial densities and their functional properties appear as a critical process in defining and shifting thermal tolerance windows . The finding of an oxygen limited thermal tolerance owing to loss of aerobic scope is in line with Taylor's and Weibel's concept of symmorphosis, which implies that excess capacity of any component of the oxygen delivery system is avoided . The present study suggests that the capacity of oxygen delivery is set to a level just sufficient to meet maximum oxygen demand between the average highs and lows of environmental temperatures . At more extreme temperatures only time limited passive survival is supported by anaerobic metabolism or the protection of molecular functions by heat shock proteins and antioxidative defence . As a corollary, the first line of thermal sensitivity is due to capacity limitations at a high level of organisational complexity, i.e . the integrated function of the oxygen delivery system, before individual, molecular or membrane functions become disturbed . These interpretations are in line with the more general consideration that, as a result of the high level of complexity of metazoan organisms compared with simple eukaryotes and then prokaryotes, thermal tolerance is reduced in metazoans . A similar sequence of sensitivities prevails within the metazoan organism, with the highest sensitivity at the organismic level and wider tolerance windows at lower levels of complexity . However, the situation is different in that loss in aerobic scope and progressive hypoxia at the organismic level define the onset of thermal limitation which then transfers to lower hierarchical levels and causes cellular and molecular disturbances . Oxygen limitation contributes to oxidative stress and, finally, denaturation or malfunction of molecular repair, e.g . during suspension of protein synthesis . The sequence of thermal tolerance limits turns into a hierarchy, ranging from systemic to cellular to molecular levels.

FEBS Lett, 2002 Jul 3, 522(1-3), 173 - 6
Identification of the E2-binding residues in the N-terminal domain of E1 of a prokaryotic pyruvate dehydrogenase complex; Hengeveld AF et al.; Pyruvate dehydrogenase (E1p) is one of the components of the pyruvate dehydrogenase multienzyme complex (PDHC) . Previously, it was shown that the N-terminal domain of E1p is involved in its binding to the core component (E2p) of PDHC . We constructed point mutations in this domain (D17Q, D17R, E20Q, E20R, D24Q and D24R) to identify the specific residues involved in these interactions . Kinetic and binding studies show that D17 is essential for the binding of E1p to E2p . D24 is involved in the binding, but not essential, whereas E20 is not involved . None of the mutations affects the folding or dimerisation of E1p.

J Mol Biol, 2002 Jul 19, 320(4), 801 - 12
Solution structure of the pro-hormone convertase 1 pro-domain from Mus musculus; Tangrea MA et al.; The solution structure of the mouse pro-hormone convertase (PC) 1 pro-domain was determined using heteronuclear NMR spectroscopy and is the first structure to be obtained for any of the domains in the convertase family . The ensemble of NMR-derived structures shows a well-ordered core consisting of a four-stranded antiparallel beta-sheet with two alpha-helices packed against one side of this sheet . Sequence homology suggests that the other eukaryotic PC pro-domains will have the same overall fold and most of the residues forming the hydrophobic core of PC1 are highly conserved within the PC family . However, some of the core residues are predicted by homology to be replaced by polar amino acid residues in other PC pro-domains and this may help to explain their marginal stability . Interestingly, the folding topology observed here is also seen for the pro-domain of bacterial subtilisin despite little or no sequence homology . Both the prokaryotic and eukaryotic structures have hydrophobic residues clustered on the solvent-accessible surface of their beta-sheets although the individual residue types differ . In the bacterial case this region is buried at the binding interface with the catalytic domain and, in the eukaryotic PC family, these surface residues are conserved . We therefore propose that the hydrophobic patch in the PC1 pro-domain is involved in the binding interface with its cognate catalytic domain in a similar manner to that seen for the bacterial system . The PC1 pro-domain structure also reveals potential mechanisms for the acid-induced dissociation of the complex between pro- and catalytic domains . (c) 2002 Elsevier Science Ltd.

J Mol Biol, 2002 Jul 19, 320(4), 783 - 99
Structural basis for mobility in the 1.1 A crystal structure of the NG domain of Thermus aquaticus Ffh; Ramirez UD et al.; The NG domain of the prokaryotic signal recognition protein Ffh is a two-domain GTPase that comprises part of the prokaryotic signal recognition particle (SRP) that functions in co-translational targeting of proteins to the membrane . The interface between the N and G domains includes two highly conserved sequence motifs and is adjacent in sequence and structure to one of the conserved GTPase signature motifs . Previous structural studies have shown that the relative orientation of the two domains is dynamic . The N domain of Ffh has been proposed to function in regulating the nucleotide-binding interactions of the G domain . However, biochemical studies suggest a more complex role for the domain in integrating communication between signal sequence recognition and interaction with receptor . Here, we report the structure of the apo NG GTPase of Ffh from Thermus aquaticus refined at 1.10 A resolution . Although the G domain is very well ordered in this structure, the N domain is less well ordered, reflecting the dynamic relationship between the two domains previously inferred . We demonstrate that the anisotropic displacement parameters directly visualize the underlying mobility between the two domains, and present a detailed structural analysis of the packing of the residues, including the critical alpha4 helix, that comprise the interface . Our data allows us to propose a structural explanation for the functional significance of sequence elements conserved at the N/G interface . (c) 2002 Elsevier Science Ltd.

Folia Microbiol (Praha), 2002, 47(3), 241 - 5
Microorganisms in a high altitude glacier ice in Tibet; Zhang XJ et al.; Eighty-one strains of viable microorganisms were recovered from 23 samples collected from Ice Core 3 of Malan Glacier (China, 91 degrees 45.3' E, 35 degrees 48.4' N) drilled at high altitude (5620 m) . All the strains were prokaryotes--75 of bacteria (including spore-forming ones) and 6 of actinomycetes . The characteristic genera differ from those of Arctic and Antarctic ice, in which many fungi and algae are widely distributed; this shows an difference of environmental conditions between Tibet and polar regions . The variation in number and species of Bacillus in different ice core layers implied changes of environmental conditions in the past.

EMBO J, 2002 Jul 1, 21(13), 3546 - 56
Structures of the pleiotropic translational regulator Hfq and an Hfq-RNA complex: a bacterial Sm-like protein; Schumacher MA et al.; In prokaryotes, Hfq regulates translation by modulating the structure of numerous RNA molecules by binding preferentially to A/U-rich sequences . To elucidate the mechanisms of target recognition and translation regulation by Hfq, we determined the crystal structures of the Staphylococcus aureus Hfq and an Hfq-RNA complex to 1.55 and 2.71 A resolution, respectively . The structures reveal that Hfq possesses the Sm-fold previously observed only in eukaryotes and archaea . However, unlike these heptameric Sm proteins, Hfq forms a homo-hexameric ring . The Hfq-RNA structure reveals that the single-stranded hepta-oligoribonucleotide binds in a circular conformation around a central basic cleft, whereby Tyr42 residues from adjacent subunits stack with six of the bases, and Gln8, outside the Sm motif, provides key protein-base contacts . Such binding suggests a mechanism for Hfq function.

Clin Diagn Lab Immunol, 2002 Jul, 9(4), 808 - 11
Antigenicity of Leishmania braziliensis histone H1 during cutaneous leishmaniasis: localization of antigenic determinants; Carmelo E et al.; The humoral immune response against Leishmania braziliensis histone H1 by patients with cutaneous leishmaniasis is described . For this purpose, the protein was purified as a recombinant protein in a prokaryotic expression system and was assayed by enzyme-linked immunosorbent assay (ELISA) with a collection of sera from patients with cutaneous leishmaniasis and Chagas' disease . The assays showed that L . braziliensis histone H1 was recognized by 66% of the serum samples from patients with leishmaniasis and by 40% of the serum samples from patients with Chagas' disease, indicating that it acts as an immunogen during cutaneous leishmaniasis . In order to locate the linear antigenic determinants of this protein, a collection of synthetic peptides covering the L . braziliensis histone H1sequence was tested by ELISA . The experiments showed that the main antigenic determinant is located in the central region of this protein . Our results show that the recombinant L . braziliensis histone H1 is recognized by a significant percentage of serum samples from patients with cutaneous leishmaniasis, but use of this protein as a tool for the diagnosis of cutaneous leishmaniasis is hampered by the cross-reaction with sera from patients with Chagas' disease.

Science, 2002 Jun 28, 296(5577), 2407 - 10
Ongoing modification of Mediterranean Pleistocene sapropels mediated by prokaryotes; Coolen MJ et al.; Late Pleistocene organic-rich sediments (sapropels) from the eastern Mediterranean Sea harbor unknown, metabolically active chemoorganotrophic prokaryotes . As compared to the carbon-lean intermediate layers, sapropels exhibit elevated cell numbers, increased activities of hydrolytic exoenzymes, and increased anaerobic glucose degradation rates, suggesting that microbial carbon substrates originate from sapropel layers up to 217,000 years old . 16S ribosomal RNA gene analyses revealed that as-yet-uncultured green nonsulfur bacteria constitute up to 70% of the total microbial biomass . Crenarchaeota constitute a smaller fraction (on average, 16%) . A slow but significant turnover of glucose could be detected . Apparently, sapropels are still altered by the metabolic activity of green nonsulfur bacteria and crenarchaeota.

Appl Environ Microbiol, 2002 Jul, 68(7), 3198 - 205
The genetic properties of the primary endosymbionts of mealybugs differ from those of other endosymbionts of plant sap-sucking insects; Baumann L et al.; Mealybugs (Hemiptera, Coccoidea, Pseudococcidae), like aphids and psyllids, are plant sap-sucking insects that have an obligate association with prokaryotic endosymbionts that are acquired through vertical, maternal transmission . We sequenced two fragments of the genome of Tremblaya princeps, the endosymbiont of mealybugs, which is a member of the beta subdivision of the Proteobacteria . Each of the fragments (35 and 30 kb) contains a copy of 16S-23S-5S rRNA genes . A total of 37 open reading frames were detected, which corresponded to putative rRNA proteins, chaperones, and enzymes of branched-chain amino acid biosynthesis, DNA replication, protein translation, and RNA synthesis . The genome of T . princeps has a number of properties that distinguish it from the genomes of Buchnera aphidicola and Carsonella ruddii, the endosymbionts of aphids and psyllids, respectively . Among these properties are a high G+C content (57.1 mol%), the same G+C content in intergenic spaces and structural genes, and similar G+C contents of the genes encoding highly and poorly conserved proteins . The high G+C content has a substantial effect on protein composition; about one-third of the residues consist of four amino acids with high-G+C-content codons . Sequence analysis of DNA fragments containing the rRNA operon and adjacent regions from endosymbionts of several mealybug species suggested that there was a single duplication of the rRNA operon and the adjacent genes in an ancestor of the present T . princeps . Subsequently, in one mealybug lineage rpS15, one of the duplicated genes, was retained, while in another lineage it decayed . These results extend the diversity of the types of endosymbiotic associations found in plant sap-sucking insects.

Appl Environ Microbiol, 2002 Jul, 68(7), 3190 - 7
Secondary (gamma-Proteobacteria) endosymbionts infect the primary (beta-Proteobacteria) endosymbionts of mealybugs multiple times and coevolve with their hosts; Thao ML et al.; Mealybugs (Hemiptera, Coccoidea, Pseudococcidae) are plant sap-sucking insects that have within their body cavities specialized cells containing prokaryotic primary endosymbionts (P-endosymbionts) . The P-endosymbionts have the unusual property of containing within their cytoplasm prokaryotic secondary endosymbionts (S-endosymbionts) {C . D . von Dohlen, S . Kohler, S . T . Alsop, and W . R . McManus, Nature (London) 412:433-436, 2001} . Four-kilobase fragments containing 16S-23S ribosomal DNA (rDNA) were obtained from the P-endosymbionts of 22 mealybug species and the S-endosymbionts of 12 representative species . Phylogenetic analyses of the P-endosymbionts indicated that they have a monophyletic origin and are members of the beta-subdivision of the Proteobacteria; these organisms were subdivided into five different clusters . The S-endosymbionts were members of the gamma-subdivision of the Proteobacteria and were grouped into clusters similar to those observed with the P-endosymbionts . The S-endosymbiont clusters were distinct from each other and from other insect-associated bacteria . The similarity of the clusters formed by the P- and S-endosymbionts suggests that the P-endosymbionts of mealybugs were infected multiple times with different precursors of the S-endosymbionts and once the association was established, the P- and S-endosymbionts were transmitted together . The lineage consisting of the P-endosymbionts of mealybugs was given the designation "Candidatus Tremblaya" gen . nov., with a single species, "Candidatus Tremblaya princeps" sp . nov . The results of phylogenetic analyses of mitochondrial DNA fragments encoding cytochrome oxidase subunits I and II from four representative mealybug species were in agreement with the results of 16S-23S rDNA analyses, suggesting that relationships among strains of "Candidatus T . princeps" are useful in inferring the phylogeny of their mealybug hosts.

Nucleic Acids Res, 2002 Jul 1, 30(13), 2886 - 93
Co-expression pattern from DNA microarray experiments as a tool for operon prediction; Sabatti C et al.; The prediction of operons, the smallest unit of transcription in prokaryotes, is the first step towards reconstruction of a regulatory network at the whole genome level . Sequence information, in particular the distance between open reading frames, has been used to predict if adjacent Escherichia coli genes are in an operon . While appreciably successful, these predictions need to be validated and refined experimentally . As a growing number of gene expression array experiments on E.coli became available, we investigated to what extent they could be used to improve and validate these predictions . To this end, we examined a large collection of published microarry data . The correlation between expression ratios of adjacent genes was used in a Bayesian classification scheme to predict whether the genes are in an operon or not . We found that for the genes whose expression levels change significantly across the experiments in the data set, the currently available gene expression data allowed a significant refinement of the sequenced-based predictions . We report these co-expression correlations in an E.coli genomic map . For a significant portion of gene pairs, however, the set of array experiments considered did not contain sufficient information to determine whether they are in the same transcriptional unit . This is not due to unreliability of the array data per se, but to the design of the experiments analyzed . In general, experiments that perturb a large number of genes offer more information for operon prediction than confined perturbations . These results provide a rationale for conducting expression studies comparing conditions that cause global changes in gene expression.

Free Radic Biol Med, 2002 Jul 1, 33(1), 1 - 14
Biological consequences of free radical-damaged DNA bases; Wallace SS; The principal oxidized cytosine bases, uracil glycol, 5-hydroxycytosine, and 5-hydroxyuracil, are readily bypassed, miscode, and are thus important premutagenic lesions . Similarly the principal oxidation product of guanine, 8-oxoguanine, miscodes with A and is a premutagenic lesion . Most of the thymine and adenine products that retain their ring structure primarily pair with their cognate bases and are not potent premutagenic lesions . Although thymine glycol pairs with its cognate base and is not mutagenic it significantly distorts the DNA molecule and is a lethal lesion . Ring fragmentation, ring contraction, and ring open products of both pyrimidines and purines block DNA polymerases and are potentially lethal lesions . Although these breakdown products have the potential to mispair during translesion synthesis, the mutational spectra of prokaryotic mutants defective in the pyrimidine-specific and/or purine-specific DNA glycosylases do not reflect that expected of the breakdown products . Taken together, the data suggest that the principal biological consequences of endogenously produced and unrepaired free radical-damaged DNA bases are mutations.

Cell, 2002 Jun 14, 109(6), 781 - 91
A mechanism of regulating transmembrane potassium flux through a ligand-mediated conformational switch; Roosild TP et al.; The regulation of cation content is critical for cell growth . However, the molecular mechanisms that gate the systems that control K+ movements remain unclear . KTN is a highly conserved cytoplasmic domain present ubiquitously in a variety of prokaryotic and eukaryotic K+ channels and transporters . Here we report crystal structures for two representative KTN domains that reveal a dimeric hinged assembly . Alternative ligands NAD+ and NADH block or vacate, respectively, the hinge region affecting the dimer's conformational flexibility . Conserved, surface-exposed hydrophobic patches that become coplanar upon hinge closure provide an assembly interface for KTN tetramerization . Mutational analysis using the KefC system demonstrates that this domain directly interacts with its respective transmembrane constituent, coupling ligand-mediated KTN conformational changes to the permease's activity.

Curr Top Microbiol Immunol, 2002, 268, 23 - 41
The 20S proteasome; Zwickl P; In contrast to our detailed knowledge of prokaryotic proteasomes, we have only a limited understanding of the prokaryotic regulators and their functional interaction with the proteasome . Most probably, we will soon learn more about the molecular structure and the mechanism of action of the prokaryotic regulators . Nevertheless, it still remains to be unravelled which signals or/and modifications transform an endogenous prokaryotic protein into a substrate of the proteasomal degradation machinery.

Mol Biol Evol, 2002 Jul, 19(7), 1066 - 82
Phylogenetic and expression analysis of the glutamate-receptor-like gene family in Arabidopsis thaliana; Chiu JC et al.; The ionotropic glutamate receptor (iGluR) gene family has been widely studied in animals and is determined to be important in excitatory neurotransmission and other neuronal processes . We have previously identified ionotropic glutamate receptor-like genes (GLRs) in Arabidopsis thaliana, an organism that lacks a nervous system . Upon the completion of the Arabidopsis genome sequencing project, a large family of GLR genes has been uncovered . A preliminary phylogenetic analysis divides the AtGLR gene family into three clades and is used as the basis for the recently established nomenclature for the AtGLR gene family . We performed a phylogenetic analysis with extensive annotations of the iGluR gene family, which includes all 20 Arabidopsis GLR genes, the entire iGluR family from rat (except NR3), and two prokaryotic iGluRs, Synechocystis GluR0 and Anabaena GluR . Our analysis supports the division of the AtGLR gene family into three clades and identifies potential functionally important amino acid residues that are conserved in both prokaryotic and eukaryotic iGluRs as well as those that are only conserved in AtGLRs . To begin to investigate whether the three AtGLR clades represent different functional classes, we performed the first comprehensive mRNA expression analysis of the entire AtGLR gene family . On the basis of RT-PCR, all AtGLRs are expressed genes . The three AtGLR clades do not show distinct clade-specific organ expression patterns . All 20 AtGLR genes are expressed in the root . Among them, five of the nine clade-II genes are root-specific in 8-week-old Arabidopsis plants.

Plant Mol Biol, 2002 Aug, 49(6), 633 - 44
AtHVA22 gene family in Arabidopsis: phylogenetic relationship, ABA and stress regulation, and tissue-specific expression; Chen CN et al.; HVA22 is an ABA- and stress-inducible gene first isolated from barley (Hordeum vulgare L.) . Homologues of HVA22 have been found in plants, animals, fungi and protozoa, but not in prokaryotes, suggesting that HVA22 plays a unique role in eukaryotes . Five HVA22 homologues, designated AtHVA22a, b, c, d and e, have been identified in Arabidopsis . These five AtHVA22 homologues can be separated into two subfamilies, with AtHVA22a, b and c grouped in one subfamily and AtHVA22d and e in the other . Phylogenetic analyses show that AtHVA22d and e are closer to barley HVA22 than to AtHVA22a, b and c, suggesting that the two subfamilies had diverged before the divergence of monocots and dicots . The distribution and size of exons of AtHVA22 homologues and barley HVA22 are similar, suggesting that these genes are descendents of a common ancestor . AtHVA22 homologues are differentially regulated by ABA, cold, dehydration and salt stresses . These four treatments enhance AtHVA22a, d and e expression, but have little or even suppressive effect on AtHVA22c expression . ABA and salt stress induce AtHVA22b expression, but cold stress suppresses ABA induction of this gene . Expression of AtHVA22d is the most tightly regulated by these four treatments among the five homologues . In general, AtHVA22 homologues are expressed at a higher level in flower buds and inflorescence stems than in rosette and cauline leaves . The expression level of these homologues in immature siliques is the lowest among all tissues analyzed . It is suggested that some of these AtHVA22 family members may play a role in stress tolerance, and others are involved in plant reproductive development.

Genomics, 2002 Jul, 80(1), 45 - 53
Novel vertebrate genes and putative regulatory elements identified at kidney disease and NR2E1/fierce loci; Abrahams BS et al.; Fierce (frc) mice are deleted for nuclear receptor 2e1 (Nr2e1), and exhibit cerebral hypoplasia, blindness, and extreme aggression . To characterize the Nr2e1 locus, which may also contain the mouse kidney disease (kd) allele, we compared sequence from human, mouse, and the puffer fish Fugu rubripes . We identified a novel gene, c222389, containing conserved elements in noncoding regions . We also discovered a novel vertebrate gene conserved across its length in prokaryotes and invertebrates . Based on a dramatic upregulation in lactating breast, we named this gene lactation elevated-1 (LACE1) . Two separate 100-bp elements within the first NR2E1 intron were virtually identical between the three species, despite an estimated 450 million years of divergent evolution . These elements represent strong candidates for functional NR2E1 regulatory elements in vertebrates . A high degree of conservation across NR2E1 combined with a lack of interspersed repeats suggests that an array of regulatory elements embedded within the gene is required for proper gene expression.

Acta Crystallogr D Biol Crystallogr, 2002 Jul, 58(Pt 7), 1211 - 3 Epub 2002 Jun 20.
Expression, purification, crystallization and preliminary X-ray analysis of rat ecto-ADP-ribosyltransferase 2 (ART2.2); Mueller-Dieckmann C et al.; ADP-ribosyltransferases catalyze the transfer of the ADP-ribose moiety from NAD(+) onto proteins and other targets . These enzymes have been found in prokaryotes and in vertebrates; a eukaryotic enzyme structure is not yet known . The enzyme from Rattus norvegicus was expressed in the Escherichia coli periplasm at a level of about 0.2 mg per litre of culture, purified and crystallized . Native data sets were collected to 2.0 A resolution . A self-rotation function revealed a local twofold axis in crystal form A and a Patterson function showed a translational relationship in form B . Form C contains only one molecule in the asymmetric unit.

J Immunol, 2002 Jul 1, 169(1), 277 - 85
Key residues of a major cytochrome P4502D6 epitope are located on the surface of the molecule; Ma Y et al.; Eukaryotically expressed CYP2D6 is the universal target of liver kidney microsomal Ab type 1 (LKM1) in both type 2 autoimmune hepatitis (AIH) and chronic hepatitis C virus (HCV) infection . In contrast, reactivity to prokaryotically expressed CYP2D6 protein and synthetic peptides is significantly lower in HCV infection than in AIH . The aim of the present study was to characterize LKM1 reactivity against a panel of eukaryotically expressed CYP2D6 constructs in the two conditions . LKM1-positive sera obtained from 16 patients with AIH and 16 with HCV infection were used as probes to perform a complete epitope mapping of CYP2D6 . Reactivity to the full-length protein and 16 constructs thereof was determined by radioligand assay . We found that antigenicity is confined to the portion of the molecule C-terminal of aa 193, no reactivity being detectable against the aa sequence 1-193 . Reactivity increases stepwise toward the C-terminal in both AIH and HCV, but the frequency of reactivity in the two conditions differs significantly between aa 267-337 . To further characterize this region, we introduced a five and a three amino acid swap mutation selected from the homologous regions of CYP2C9 and HCV . This maneuver resulted in a substantial loss of LKM1 binding in both conditions, suggesting that this region contains a major epitope . Molecular modeling revealed that CYP2D6(316-327) is exposed on the surface of the protein, and may represent a key target for the autoantibody . These findings provide an initial characterization of the antigenic constitution of the target of LKM1 in AIH and HCV infection.

FEMS Microbiol Lett, 2002 Jun 4, 211(2), 175 - 82
Use of subtractive hybridization for comprehensive surveys of prokaryotic genome differences; Agron PG et al.; Comparative bacterial genomics shows that even different isolates of the same bacterial species can vary significantly in gene content . An effective means to survey differences across whole genomes would be highly advantageous for understanding this variation . Here we show that suppression subtractive hybridization (SSH) provides high, representative coverage of regions that differ between similar genomes . Using Helicobacter pylori strains 26695 and J99 as a model, SSH identified approximately 95% of the unique open reading frames in each strain, showing that the approach is effective . Furthermore, combining data from parallel SSH experiments using different restriction enzymes significantly increased coverage compared to using a single enzyme . These results suggest a powerful approach for assessing genome differences among closely related strains when one member of the group has been completely sequenced.

Adv Microb Physiol, 2002, 46, 257 - 318
Biochemistry, regulation and genomics of haem biosynthesis in prokaryotes; O'Brian MR et al.; Haems are involved in many cellular processes in prokaryotes and eukaryotes . The biosynthetic pathway leading to haem formation is, with few exceptions, well-conserved, and is controlled in accordance with cellular function . Here, we review the biosynthesis of haem and its regulation in prokaryotes . In addition, we focus on a modification of haem for cytochrome c biogenesis, a complex process that entails both transport between cellular compartments and a specific thioether linkage between the haem moiety and the apoprotein . Finally, a whole genome analysis from 63 prokaryotes indicates intriguing exceptions to the universality of the haem biosynthetic pathway and helps define new frontiers for future study.

Vet Immunol Immunopathol, 2002 Sep 10, 87(3-4), 215 - 21
Modulating immune responses with dendritic cells: an attainable goal in veterinary medicine?
Vecchione A, Catchpole B, D'Mello F, Kanellos T, Hamblin A.
Dendritic cells (DCs) are antigen presenting cells that potently modulate immune responses with varying outcomes depending on the DC sub-population involved . To understand how DC sub-types arise, it is necessary to determine which factors influence their differentiation . At least three major sub-populations of DCs have been described in mice: CD4+/CD8- "myeloid" DCs, CD4-/CD8+ "lymphoid" DCs and Langerhans cell-derived DCs . Whilst somewhat comparable populations have been described in man, in most other species very little is known . The identification of cytokines which stimulate proliferation of DC precursors, and the observation that the cytokine environment influences the phenotype and the function of the DCs that subsequently develop, has provided a useful tool for evaluating these rare cells . We describe the influence of cytokines on the phenotype of DCs generated in the rat . Using bone marrow cells as the source of precursors we generated "myeloid-type" DCs from the adherent population using granulocyte-macrophage colony stimulating factor (GM-CSF), IL-4 and Flt-3L or "lymphoid-type" DCs from the non-adherent population using cytokines which included IL-7, IL-3, SCF and TNFalpha . In order to facilitate similar approaches to the study of equine DCs we have identified the nucleotide sequence encoding GM-CSF from the m-RNA of equine PBMC stimulated with Concanavalin A, amplified the cDNA by PCR and cloned it in eukaryotic and prokaryotic expression vectors . We report on the structure and function of this molecule.

Environ Microbiol, 2002 Jun, 4(6), 338 - 48
Changes in archaeal, bacterial and eukaryal assemblages along a salinity gradient by comparison of genetic fingerprinting methods in a multipond solar saltern; Casamayor EO et al.; Microbial communities inhabiting a multipond solar saltern were analysed and compared using SSU rRNA polymerase chain reaction (PCR)-based fingerprintings carried out in parallel by four laboratories . A salinity gradient from seawater (3.7%) to NaCl precipitation (37%) was studied for Bacteria, Archaea and Eukarya, and laboratories applied their own techniques and protocols on the same set of samples . Members of all three domains were retrieved from all salt concentrations . Three fingerprinting techniques were used: denaturing gradient gel electrophoresis (DGGE), ribosomal internal spacer analysis (RISA), and terminal-restriction fragments length polymorphism (T-RFLP) . In addition, each laboratory used its own biomass collection method and DNA extraction protocols . Prokaryotes were addressed using DGGE and RISA with different 'domain-specific' primers sets . Eukaryotes were analysed by one laboratory using DGGE and T-RFLP, but targeting the same 18S rDNA site . Fingerprints were compared through cluster analysis and non-metric multidimensional scaling plots . This exercise allowed fast comparison of microbial assemblages and determined to what extent the picture provided by each laboratory was similar to those of others . Formation of two main, salinity-based groups of samples in prokaryotes (4-15% and 22-37% salinity) was consistent for all the laboratories . When other clusters appeared, this was a result of the particular technique and the protocol used in each case, but more affected by the primers set used . Eukaryotic microorganisms changed more from pond to pond; 4-5% and 8-37% salinity were but the two main groups detected . Archaea showed the lowest number of bands whereas Eukarya showed the highest number of operational taxonomic units (OTUs) in the initial ponds . Artefacts appeared in the DGGE from ponds with extremely low microbial richness . On the other hand, different 16S rDNA fragments with the same restriction or internal transcribed spacer (ITS) length were the main limitations for T-RFLP and RISA analyses, respectively, in ponds with the highest OTUs richness . However, although the particular taxonomic composition could vary among protocols, the general structure of the microbial assemblages was maintained.

Genetika, 2002 May, 38(5), 695 - 701
{On the problem of genome redundancy in viruses and prokaryotes}; Sadovskii MG; A specific index of nucleotide sequence redundancy, the specific restriction length of a finite frequency dictionary, was determined for a complete set of genes in some viral genomes and a genome of a bacterium, Bacillus subtilis . The distribution of the gene number over the specific restriction length was shown to be bimodal for viral genomes and unimodal for the Bac . subtilis genome . These results agree with earlier data.

Plant Physiol, 2002 Jun, 129(2), 691 - 705
RASPBERRY3 gene encodes a novel protein important for embryo development; Apuya NR et al.; We identified a new gene that is interrupted by T-DNA in an Arabidopsis embryo mutant called raspberry3 . raspberry3 has "raspberry-like" cellular protuberances with an enlarged suspensor characteristic of other raspberry embryo mutants, and is arrested morphologically at the globular stage of embryo development . The predicted RASPBERRY3 protein has domains found in proteins present in prokaryotes and algae chloroplasts . Computer prediction analysis suggests that the RASPBERRY3protein may be localized in the chloroplast . Complementation analysis supports the possibility that the RASPBERRY3 protein may be involved in chloroplast development . Our experiments demonstrate the important role of the chloroplast, directly or indirectly, in embryo morphogenesis and development.

Plant Physiol, 2002 Jun, 129(2), 500 - 15
Two-component signal transduction pathways in Arabidopsis; Hwang I et al.; The two-component system, consisting of a histidine (His) protein kinase that senses a signal input and a response regulator that mediates the output, is an ancient and evolutionarily conserved signaling mechanism in prokaryotes and eukaryotes . The identification of 54 His protein kinases, His-containing phosphotransfer proteins, response regulators, and related proteins in Arabidopsis suggests an important role of two-component phosphorelay in plant signal transduction . Recent studies indicate that two-component elements are involved in plant hormone, stress, and light signaling . In this review, we present a genome analysis of the Arabidopsis two-component elements and summarize the major advances in our understanding of Arabidopsis two-component signaling.

J Appl Microbiol, 2002, 93(1), 15 - 28
Salt stress induces programmed cell death in prokaryotic organism Anabaena; Ning SB et al.; AIMS: Our main interest is to check if programmed cell death (PCD) can occur in prokaryotic algae and if the morphological and biochemical features of PCD are conserved . METHODS AND RESULTS: Using TUNEL labelling, fluorescence and light microscopy and DNA gel electrophoresis, we found that cell death with features similar to those in metazoan PCD could be induced in different Anabaena strains after exposure to univalent-cation salts at moderate concentration . These features included specific DNA fragmentation, cytoplasmic vacuolation, and the progressive disorganization, fragmentation and subsequent autolysis of the cell corpse . Further analyses of cell viability and proteinase activity revealed that increased protease activities, decreased DNA content, and loss of plasmalemma integrity were related to the PCD process . CONCLUSIONS: The results showed that like PCD in eukaryotes, PCD in Anabaena is an active process, and is an adaptation to adverse environments . The features of PCD shared between eukaryotes and Anabaena suggest that PCD mechanisms are conserved during evolution . SIGNIFICANCE AND IMPACT OF THE STUDY: The results will contribute greatly to our understanding of PCD origin and evolution, and are potentially useful in controlling the deluge of algae in some lakes.

In Silico Biol, 2002, 2(2), 87 - 95
Operon conservation from the point of view of Escherichia coli, and inference of functional interdependence of gene products from genome context; Moreno-Hagelsieb G et al.; We have previously demonstrated that genes within experimentally characterized operons of Escherichia coli are conserved together in other genomes more frequently than genes at the borders of transcription units . Here we expand the analyses and show that, as the phylogenetic distance of the genomes compared increases, the genes remaining together must belong to genes associated into operons in other prokaryotes regardless of the operon organization of the corresponding orthologous gene pair of E . coli . At the same time, we show that the observed tendencies of genes within operons to keep very short inter-genic distances in E . coli, is the same in any other prokaryote whose genome is currently available . We also show the relationship between our analyses of conservation and the inference of functional relationships from genomic context.

Infect Immun, 2002 Jul, 70(7), 3923 - 9
Essential role of ferritin Pfr in Helicobacter pylori iron metabolism and gastric colonization; Waidner B et al.; The reactivity of the essential element iron necessitates a concerted expression of ferritins, which mediate iron storage in a nonreactive state . Here we have further established the role of the Helicobacter pylori ferritin Pfr in iron metabolism and gastric colonization . Iron stored in Pfr enabled H . pylori to multiply under severe iron starvation and protected the bacteria from acid-amplified iron toxicity, as inactivation of the pfr gene restricted growth of H . pylori under these conditions . The lowered total iron content in the pfr mutant, which is probably caused by decreased iron uptake rates, was also reflected by an increased resistance to superoxide stress . Iron induction of Pfr synthesis was clearly diminished in an H . pylori feoB mutant, which lacked high-affinity ferrous iron transport, confirming that Pfr expression is mediated by changes in the cytoplasmic iron pool and not by extracellular iron . This is well in agreement with the recent discovery that iron induces Pfr synthesis by abolishing Fur-mediated repression of pfr transcription, which was further confirmed here by the observation that iron inhibited the in vitro binding of recombinant H . pylori Fur to the pfr promoter region . The functions of H . pylori Pfr in iron metabolism are essential for survival in the gastric mucosa, as the pfr mutant was unable to colonize in a Mongolian gerbil-based animal model . In summary, the pfr phenotypes observed give new insights into prokaryotic ferritin functions and indicate that iron storage and homeostasis are of extraordinary importance for H . pylori to survive in its hostile natural environment.

EMBO J, 2002 Jun 17, 21(12), 3119 - 27
Prokaryotic DNA segregation by an actin-like filament; Moller-Jensen J et al.; The mechanisms responsible for prokaryotic DNA segregation are largely unknown . The partitioning locus (par) encoded by the Escherichia coli plasmid R1 actively segregates its replicon to daughter cells . We show here that the ParM ATPase encoded by par forms dynamic actin-like filaments with properties expected for a force-generating protein . Filament formation depended on the other components encoded by par, ParR and the centromere-like parC region to which ParR binds . Mutants defective in ParM ATPase exhibited hyperfilamentation and did not support plasmid partitioning . ParM polymerization was ATP dependent, and depolymerization of ParM filaments required nucleotide hydrolysis . Our in vivo and in vitro results indicate that ParM polymerization generates the force required for directional movement of plasmids to opposite cell poles and that the ParR-parC complex functions as a nucleation point for ParM polymerization . Hence, we provide evidence for a simple prokaryotic analogue of the eukaryotic mitotic spindle apparatus.

EMBO J, 2002 Jun 17, 21(12), 3108 - 18
Cell cycle-dependent localization of two novel prokaryotic chromosome segregation and condensation proteins in Bacillus subtilis that interact with SMC protein; Mascarenhas J et al.; Disruption of ypuG and ypuH open reading frames in Bacillus subtilis leads to temperature-sensitive slow growth, a defect in chromosome structure and formation of anucleate cells . The genes, which were named scpA and scpB, were found to be epistatic to the smc gene . Fusions of ScpA and ScpB to the fluorescent proteins YFP or CFP showed that both proteins co-localize to two or four discrete foci that were present at mid-cell in young cells, and within both cell halves, generally adjacent to chromosomal origin regions, in older cells . ScpA and ScpB foci are associated with DNA and depend on the presence of SMC and both Scps . ScpA and ScpB are associated with each other and with SMC in vivo, as determined using the FRET technique and immunoprecipitation assays . Genes similar to scpA and scpB are present in many bacteria and archaea, which suggests that their gene products form a condensation complex with SMC in most prokaryotes . The observed foci could constitute condensation factories that pull DNA away from mid-cell into both cell halves.

Cytogenet Cell Genet, 2001, 95(3-4), 146 - 52
Localization, genomic organization, and alternative transcription of a novel human SAM-dependent methyltransferase gene on chromosome 2p22-->p21; Zhang Y et al.; As part of our studies to identify the gene responsible for hereditary gingival fibromatosis, GINGF (OMIM 135300), we have identified and cloned a novel human gene that contains the highly conserved methyltransferase domain characteristic of S-adenosylmethionine-dependent methyltransferases . We localized this gene (C2orf8 encoding 288L6 SAM-methyltransferase) to chromosome 2p22-->p21 by FISH, and sublocalized it to BAC RP11 288L6 flanked by D2S2238 and D2S2331 . Computational analysis of aligned ESTs identified ten exons in the hypothetical C2orf8 gene . Results of RACE analyses in placenta identified multiple transcripts of this gene with heterogeneity at the 5'-UTR . Alternative transcription and tissue specific expression of C2orf8 were detected by RT-PCR and Northern blot analyses . C2orf8 is expressed in a variety of tissues including brain, colon, gingiva, heart, kidney, liver, lung, placenta, small intestine, spleen, and thymus . Open reading frame analysis of the alternative transcripts identified a shared coding region spanning exons 6-10 . This ORF consists of 732 nucleotides encoding a putative 244 amino acid protein . Bioinformational searches of both C2orf8 and the putative protein product identified three methyltransferase motifs conserved across many prokaryotic and eukaryotic species . Sequence analyses of C2orf8 excluded coding region mutations as causative of GINGF .

Nucleic Acids Res, 2002 Jun 15, 30(12), 2710 - 25
Dictionary-driven prokaryotic gene finding; Shibuya T et al.; Gene identification, also known as gene finding or gene recognition, is among the important problems of molecular biology that have been receiving increasing attention with the advent of large scale sequencing projects . Previous strategies for solving this problem can be categorized into essentially two schools of thought: one school employs sequence composition statistics, whereas the other relies on database similarity searches . In this paper, we propose a new gene identification scheme that combines the best characteristics from each of these two schools . In particular, our method determines gene candidates among the ORFs that can be identified in a given DNA strand through the use of the Bio-Dictionary, a database of patterns that covers essentially all of the currently available sample of the natural protein sequence space . Our approach relies entirely on the use of redundant patterns as the agents on which the presence or absence of genes is predicated and does not employ any additional evidence, e.g . ribosome-binding site signals . The Bio-Dictionary Gene Finder (BDGF), the algorithm's implementation, is a single computational engine able to handle the gene identification task across distinct archaeal and bacterial genomes . The engine exhibits performance that is characterized by simultaneous very high values of sensitivity and specificity, and a high percentage of correctly predicted start sites . Using a collection of patterns derived from an old (June 2000) release of the Swiss-Prot/TrEMBL database that contained 451 602 proteins and fragments, we demonstrate our method's generality and capabilities through an extensive analysis of 17 complete archaeal and bacterial genomes . Examples of previously unreported genes are also shown and discussed in detail.

Physiol Plant, 2002 Jun, 115(2), 221 - 227
Drought stress affects chloroplast lipid metabolism in rape (Brassica napus) leaves; Benhassaine-Kesri G et al.; Rape (Brassica napus L . var . Bienvenue) is a 16:3 plant which contains predominantly prokaryotic species of monogalactosyldiacylglycerol i.e . sn-1 C18, sn-2 C16 (C18/C16 MGDG) . Rape plants were exposed to a restricted water supply for 12 days . Under drought conditions, considerable changes in lipid metabolism were observed . Drought stress provoked a decline in leaf polar lipids, which is mainly due to a decrease in MGDG content . Determination of molecular species in phosphatidylcholine (PC) and MGDG indicated that the prokaryotic molecular species of MGDG (C18/C16) decreased after drought stress while the eukaryotic molecular species (C18/C18) remained stable . Drought stress had different effects on two key enzymes of PC and MGDG synthesis . The in vitro activity of MGDG synthase (EC . 2.4.1.46) was reduced in drought stressed plants whereas cholinephosphotransferase (EC . 2.7.8.2) activity was not affected . Altogether these results suggest that the prokaryotic pathway leading to MGDG synthesis was strongly affected by drought stress while the eukaryotic pathway was not . It was also observed that the molecular species of leaf PC became more saturated in drought stressed plants . This could be due to a specific decrease in oleate desaturase activity.

Farmaco, 2002 May, 57(5), 385 - 415
The medicinal chemistry of multidrug resistance (MDR) reversing drugs; Teodori E et al.; Multidrug resistance (MDR) is a kind of resistance of cancer cells to multiple classes of chemotherapic drugs that can be structurally and mechanistically unrelated . Classical MDR regards altered membrane transport that results in lower cell concentrations of cytotoxic drug and is related to the over expression of a variety of proteins that act as ATP-dependent extrusion pumps . P-glycoprotein (Pgp) and multidrug resistance protein (MRP1) are the most important and widely studied members of the family that belongs to the ABC superfamily of transporters . It is apparent that, besides their role in cancer cell resistance, these proteins have multiple physiological functions as well, since they are expressed also in many important non-tumoural tissues and are largely present in prokaryotic organisms . A number of drugs have been identified which are able to reverse the effects of Pgp, MRPI and sister proteins, on multidrug resistance . The first MDR modulators discovered and studied in clinical trials were endowed with definite pharmacological actions so that the doses required to overcome MDR were associated with unacceptably high side effects . As a consequence, much attention has been focused on developing more potent and selective modulators with proper potency, selectivity and pharmacokinetics that can be used at lower doses . Several novel MDR reversing agents (also known as chemosensitisers) are currently undergoing clinical evaluation for the treatment of resistant tumours . This review is concerned with the medicinal chemistry of MDR reversers, with particular attention to the drugs that are presently in development.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2000, 32(5), 499 - 502
Cloning and Optimized Expression of Human Angiogenin in E.coli; Lu F et al.; Angiogenin(ANG) is an important factor of angiogenesis during different stage of tumor development and exists widely in various tumors . To study the biological funcption and find the antagonistic drugs of angiogenin, the angiogenin was allowed to be expressed by E.coli . By the aid of computer, the sequence around the start codon of angiogenin gene was modified according to local secondary structure . The modified human ang gene was amplified by reverse-transcription polymerase chain reaction from the human lung cancer cell line A549, and inserted into the prokaryotic expression vector pLDH99 . After screening, high expression recombinants were obtained, and the expression level of the hANG was about 30% of total bacteria protein by SDS-PAGE . Biological assays indicated that the rhANG could induce new blood vessel formation in CAM in vitro . Our data showed that the recombinant hANG was active and the optimized expression of ang gene was practicable.

J Bacteriol, 2002 Jul, 184(13), 3671 - 81
Genome-wide dynamic transcriptional profiling of the light-to-dark transition in Synechocystis sp . strain PCC 6803; Gill RT et al.; We report the results of whole-genome transcriptional profiling of the light-to-dark transition with the model photosynthetic prokaryote Synechocystis sp . strain PCC 6803 (Synechocystis) . Experiments were conducted by growing Synechocystis cultures to mid-exponential phase and then exposing them to two cycles of light/dark conditions, during which RNA samples were obtained . These samples were probed with a full-genome DNA microarray (3,169 genes, 20 samples) as well as a partial-genome microarray (88 genes, 29 samples) . We concluded that (i) 30-min sampling intervals accurately captured transcriptional dynamics throughout the light/dark transition, (ii) 25% of the Synechocystis genes (783 genes) responded positively to the presence of light, and (iii) the response dynamics varied greatly for individual genes, with a delay of up to 120 to 150 min for some genes . Four classes of genes were identified on the basis of their dynamic gene expression profiles: class I (108 genes, 30-min response time), class II (279 genes, 60 to 90 min), class III (258 genes, 120 to 150 min), and class IV (138 genes, 180 min) . The dynamics of several transcripts from genes involved in photosynthesis and primary energy generation are discussed . Finally, we applied Fisher discriminant analysis to better visualize the progression of the overall transcriptional program throughout the light/dark transition and to determine those genes most indicative of the lighting conditions during growth.

Biochemistry, 2002 Jun 18, 41(24), 7597 - 603
The role of surface-exposed lysines in wrapping DNA about the bacterial histone-like protein HU; Grove A et al.; Several basic proteins, including the ubiquitous HU proteins, serve histone-like functions in prokaryotes . Significant sequence conservation exists between HU homologues; yet binding sites varying from 9 to 37 bp have been reported . TF1, an HU homologue with a 37 bp binding site that is encoded by the Bacillus subtilis bacteriophage SPO1, binds with nM affinity to DNA that contains 5-hydroxymethyluracil (hmU) in place of thymine and to T-containing DNA with loops . We evaluated the contribution of three conserved lysines to specifying the length of the binding site and show that Lys3 is critical for maintaining a long binding site in T-containing DNA: A mutant protein in which Lys3 is replaced with Gln(TF1-K3Q) is completely deficient in forming a stable complex . The affinity for 37 bp hmU-containing DNA is also reduced, from approximately 3 nM for wild-type TF1 to approximately 90 nM for TF1-K3Q . The decrease in affinity of TF1-K3Q for hmU-containing DNA > or = 25 bp suggests that Lys3 contacts DNA 8-9 bp distal to the sites of kinking . We propose that Lys3 forms an internal saltbridge to Asp26 in HU homologues characterized by shorter binding sites and that its surface exposure, and hence a longer binding site, may correlate with absence of this aspartate.

Microbiology, 2002 Jun, 148(Pt 6), 1931 - 7
The lon gene, encoding an ATP-dependent protease, is a novel member of the HAIR/HspR stress-response regulon in actinomycetes; Sobczyk A et al.; Members of a family of ATP-dependent proteases related to Lon from Escherichia coli are present in most prokaryotes and eukaryotes . These proteases are generally reported to be heat induced, and various regulatory systems have been described . The authors cloned and disrupted the lon gene and studied the regulation of its expression in Streptomyces lividans . lon is negatively regulated by the HspR/HAIR repressor/operator system, suggesting that Lon is produced concomitantly with the other members of this regulon, DnaK and ClpB . The lon mutant grew more slowly than the wild-type and spore germination was impaired at high temperature . Nevertheless its cell cycle was not greatly affected and it sporulated normally.

Methods, 2002 Feb, 26(2), 105 - 14
Toeprint analysis of the positioning of translation apparatus components at initiation and termination codons of fungal mRNAs; Sachs MS et al.; The ability to map the position of ribosomes and their associated factors on mRNAs is critical for an understanding of translation mechanisms . Earlier approaches to monitoring these important cellular events characterized nucleotide sequences rendered nuclease-resistant by ribosome binding . While these approaches furthered our understanding of translation initiation and ribosome pausing, the pertinent techniques were technically challenging and not widely applied . Here we describe an alternative assay for determining the mRNA sites at which ribosomes or other factors are bound . This approach uses primer extension inhibition, or "toeprinting," to map the 3' boundaries of mRNA-associated complexes . This methodology, previously used to characterize initiation mechanisms in prokaryotic and eukaryotic systems, is used here to gain an understanding of two interesting translational regulatory phenomena in the fungi Neurospora crassa and Saccharomyces cerevisiae: (a) regulation of translation in response to arginine concentration by an evolutionarily conserved upstream open reading frame, and (b) atypical termination events that occur as a consequence of the presence of premature stop codons . (c) 2002 Elsevier Science (USA).

J Mol Biol, 2002 Jun 7, 319(3), 587 - 91
The hexameric ring structure of the Escherichia coli RuvB branch migration protein; Chen YJ et al.; The RuvB protein is part of the homologous recombination machinery in prokaryotic cells . Many studies have shown that RuvB is organized into hexameric rings functioning as DNA pumps at Holliday junctions, using ATP hydrolysis to drive branch migration . Structures now exist for two RuvB proteins, as well as for several structurally homologous proteins, including the replication factor-C small subunit (RFCS) . Two models for the possible hexameric organization of RuvB subunits have been proposed, based upon the hexameric structures of NSF and HslU, two AAA-ATPases involved in vesicle fusion and proteolysis, respectively . We have used electron microscopy to generate an improved three-dimensional reconstruction of the double hexamers formed by Escherichia coli RuvB on double-stranded DNA . We find that an atomic model of the hexameric RFCS provides a significantly better fit to the RuvB hexamer than do the models for RuvB generated from NSF and HslU . This suggests that there may be a highly conserved structure for many proteins involved in different aspects of DNA replication, recombination, transcription and repair . (c) 2002 Elsevier Science Ltd.

Biochem Biophys Res Commun, 2002 May 3, 293(2), 806 - 15
Overexpression of Arabidopsis response regulators, ARR4/ATRR1/IBC7 and ARR8/ATRR3, alters cytokinin responses differentially in the shoot and in callus formation; Osakabe Y et al.; Arabidopsis ARR4/ATRR1/IBC7 and ARR8/ATRR3 are homologous genes of prokaryotic response regulators that are involved in the His-Asp phosphorelay signal transduction . We analyzed the function of these genes as response regulators using transgenic plants . Overexpression of ARR4 in cultured stems of the transgenics markedly promoted shoot formation in the presence of cytokinin, while overexpression of ARR8 repressed shoot formation and greening of calli . The expression level of cytokinin-inducible genes, cycD3 and cab increased in the ARR4 overexpressor but decreased in the ARR8 overexpressor . By contrast, two drought stress-inducible genes, rd29A and erd1, were expressed in both overexpressors as that in control plants . These results suggest that ARR4 and ARR8 are involved in cytokinin signal transduction, and that ARR4 functions as a positive-regulator, whereas ARR8 functions as a negative-regulator . Furthermore, microarray analysis showed that several genes were up-regulated in the ARR4 overexpressor . Consistent with these results, ARR4 and ARR8 might play important roles in the sensoring system of cytokinin signal transduction pathway in various developmental and environmental conditions and the regulation of gene expression .

Anal Biochem, 2002 Jun 15, 305(2), 242 - 50
A rapid screening method to monitor expression of recombinant proteins from various prokaryotic and eukaryotic expression systems using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry; Jebanathirajah JA et al.; Rapid methods using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry to monitor recombinant protein expression from various prokaryotic and eukaryotic cell culture systems were devised . Intracellular as well as secreted proteins from both induced and constitutive expression systems were measured and monitored from whole cells and growth media, thus providing an alternative to time-consuming traditional methods for screening and monitoring of protein expression . The methods described here involve minimal processing of samples and are therefore relevant to high-throughput screening applications .

FEMS Immunol Med Microbiol, 2002 Jun 3, 33(2), 77 - 88
Isolation and characterisation of a 13.8-kDa bacteriolytic enzyme from house dust mite extracts: homology with prokaryotic proteins suggests that the enzyme could be bacterially derived; Mathaba LT et al.; Bacteriolytic activity was detected in extracts of whole mite and spent growth medium (SGM) from the clinically important Dermatophagoides pteronyssinus and Dermatophagoides farinae mites and was most abundant in whole mite extract . Gram-positive organisms Micrococcus lysodeikticus, Bacillus megaterium and Listeria monocytogenes were preferentially lysed and the lytic activity was enhanced by thiols, destroyed by mite proteases, inhibited by HgCl2 and high concentrations of NaCl but was resistant to heat and acid treatment . Substrate SDS-PAGE analysis indicated the presence of several lytic enzymes, two of which were isolated from D . pteronyssinus spent growth medium extract by hydroxyapatite chromatography . The N-terminal amino acid sequence of one of them was then used in PCR-based cloning studies . The complete amino acid sequence of this protein was determined and cDNA found to encode a 130-amino acid residue mature protein with a 20-amino acid leader sequence . The deduced protein demonstrated sequence similarity with the C-terminal regions of a group of bacterial proteins belonging to the P60 superfamily . These data suggest that the enzyme is derived from bacteria within the mites rather than from mites per se.

Curr Pharm Des, 2002, 8(13), 1119 - 35
Microbial genomics and novel antibiotic discovery: new technology to search for new drugs; Dougherty TJ et al.; The process of prokaryotic drug discovery has been a model of success for over fifty years, yet the number of exploited bacterial targets is a mere fraction, less than 0.1% of the potential targets (based on total number of bacterial genes identified by gene sequence projects) . To better understand the potential for drug intervention, multiple paradigms have been established in the pharmaceutical industry, all with some semblance of commonality and uniqueness to provide proprietary positioning, yet no company has been successful to date in taking a genomics approach to the finish line of having a genomics-based drug on the market . Within this overview, we provide a strategic overview of a sample process for the identification, validation and exploitation of novel antibacterial targets ascertained through a bioinformatics-based genomics drug discovery program.

Curr Pharm Des, 2002, 8(13), 1099 - 118
Genomics in anti-infective drug discovery--getting to endgame; Haney SA et al.; Whole chromosome sequence of prokaryotes has provided the availability of multiple bacterial pathogen sequence data and it has become a widely used tool in the drug discovery process . However the sequence data in itself is merely a starting point for drug discovery of novel antibacterial targets and, eventually, drugs . In order to leverage this large amount of data it is necessary to match an understanding of the microbial physiology of pathogenic bacteria to disease processes and identifying the gene products for which intervention may reduce or eliminate the infectious state . However, to date, the application of genomics to anti-infective drug discovery has not, since 1995 with the first complete sequence of a pathogenic bacterium, led to identification of a novel antibacterial agent . Here we review the field of bacterial genomics and how it is enabling the drug discovery process . Many new molecular-based technologies (proteomics, transcriptional profiling, studies of gene expression in vivo) have originated or have expanded into wider use, and have been made possible by the availability of complete bacterial genome sequence information and subsequent bioinformatic analytic tools . Taken together, these technologies, overlaid within an established drug discovery program, now affords the opportunity for the identification, validation, and process design for high-throughput target mining at unprecedented volumes and timeframes.

Fungal Genet Biol, 2002 Jun, 36(1), 35 - 46
Mutation and functional analysis of the Aspergillus nidulans ammonium permease MeaA and evidence for interaction with itself and MepA; Monahan BJ et al.; The movement of ammonium across biological membranes is mediated in both prokaryotic and eukaryotic systems by ammonium transport proteins which constitute a family of related sequences (called the AMT/MEP family) . Interestingly, recent evidence suggests that human and mouse Rhesus proteins which display significant relatedness to AMT/MEP sequences may function as ammonium transporters . To add to the functional understanding of ammonium transport proteins, the sequence changes in 37 loss-of-function mutations within the Aspergillus nidulans ammonium permease gene, meaA, were characterized . Together with the identification of conserved AMT/MEP residues and regions, the mutational analysis predicted regions important for uptake activity . Specifically, a major facilitator superfamily like motif (161-GAVAERGR-168 in MeaA) may be important for the translocation of ammonium across the membrane as may the conserved Pro186 residue . A specific Gly447 to Asp mutation was introduced into MeaA and this mutant protein was found to trans-inhibit the activity of endogenous MeaA and the other A . nidulans ammonium transporter, MepA . These results suggest that MeaA may interact with itself and with MepA, although any hetero-interaction is not required for ammonium transport function . In addition, cross-feeding studies showed that MeaA and to a lesser extent MepA are also required for the retention of intracellular ammonium.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2001, 33(2), 229 - 232
Small Subunit Ribosomal RNA Genes of Microsporidia; Wang JY et al.; Microsporidia are ubiquitous intracellular parasitic protozoa infecting all types of animals . Their ribosomes and rRNAs are of prokaryotic size.In order to better understand their phylogenetic relationship and identify the uncertain species, the sequences of the small subunit ribosomal RNA (ssurRNA, 16 S rRNA) genes of nine microsporidia infectious to the silkworm, Bombyx mori, were determined . The results of phylogenetic trees and Southern blotting suggest all the nine strains of microsporidia are various species of the genus Nosema.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2001, 33(2), 225 - 228
Expression, Purification and in vitro Activity of Apoptin; Sun GJ et al.; A novel protein named apoptin induces a p53-independent, Bcl-2 insensitive type apoptosis in various human tumor cells but not in normal cells . Apoptin is considered to be a promising candidate for safe and effective anti-tumortherapy . Moreover, apoptin may be important for fundamental studies on molecularbasis of apoptosis and cancer . Apoptin gene was subcloned into prokaryotic expression vector pPROEXHT and pBV220, respectively . The apoptin protein was obtained from pPROEXHT-apoptin expression system and not from pBV220-apoptin the former contains a 6xhistidine affinity tag for ease of purification . Expressed protein was purified by chromatography on a co-chelated affinity column and was renatured by dialysis . The renatured apoptin was able to induce purified Hela nuclei apoptosis in vitro even without the participation of the cytoplasm, showing that apoptin expressed in E.coli was active to induce apoptosis.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2001, 33(2), 210 - 214
Cloning, Expression, Purification and Activity of the hsBLyS; Liu P et al.; The full length cDNA of human B lymphocyte stimulator (hBLyS) was amplified by using PCR method from cDNA library of human placenta . After purifying and sequencing, the DNA fragment of functional domain of hBLyS (hsDNA fragment)was amplified by using nested PCR method from the PCR product . The prokaryotic expression plasmid pET-30a( )/ hBLyS was constructed with recombinant DNA techniques after purifying and identifying the hsDNA fragment . Then the plasmid pET-30a( )/ hBLyS was transformed into lambdaDE3 cells and the recombination protein was found to be highly expressed the expression product was purified by affinity chromatography gel, Ni(2 )-IDA, made in our laboratory . The experimental results showed that the sequence of the PCR product was identical with the published hBLyS cDNA sequence and purity of the recombination protein we obtained was high . The activity of the purified recombination protein was very significant in the proliferation test of B lymphocytes.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2001, 33(3), 360 - 364
A Candidate Oral Vaccine to Helicobacter pylori Fusion Protein of HspA and CtxB; Li MF et al.; Heat-shock protein A subunit(HspA), an effective immunogen may stimulate the immunoresponse in human body against challenge of H.pylori . The B subunit of cholera toxin (CtxB) has been proved to be a potent mucosal immunogen, actas an adjuvant for vaccine targeted for delivery to the mucosal-associated lymphoid tissue . A recombinant plasmid expressing bivalent antigen of HspA and CtxB subunit was constructed as follows . hspA and ctxB gene was amplified by PCR . The DNA products of hspA and ctxB were inserted in the prokaryotic expression vector pET-22b( ), respectively, and then the resulted recombinant plasmid expressinga fusion protein named HCT was transformed into the E.coli strain BL-21(DE3) . hct gene was measured to be 708 base pairs long, and the fusion protein encoding a polypeptide of 236 amino acid residues, corresponded to a calculated molecular masses of 30 kD . Western blot analysis of the recombinant protein HCT confirmed that it could be specifically recognized by the serum of H.pylori-infected patients . HspA and HCT labelled (125)I were orally administered into the stomach of mice, respectively, and the radioactivity of (125)I in serum of each mouse was assayed at intervals 15 min, 30 min, 60 min, 90 min and 120 min . The result indicated that there were high radioactivity counts in the groups of HCT than that of HspA(P 0.001) . This result suggests that the CtxB may enhance the volume of HspA absorbed from the intestine of mice, therefore the recombinant fusion protein HCT may be an effective oral vaccine for prevention and treatment against the infection of H.pylori.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2001, 33(3), 281 - 285
Cloning and Expression of Targeting CuZn-SOD to Central Nervous System; He HJ et al.; Oxygen-derived free radicals are thought to be involved in the pathogenesis of a wide range of neurological disorders . Targeted delivery of CuZn-SOD to neurons in central nervous system may have therapeutic value in such diseases . The gene encoding human CuZn-SOD was fused to tetanus toxin fragment C geneto construct a fusion gene, then it was cloned into prokaryotic expression vector pET-22b( ) and baculovirus vector pFastBacHTb, and was expressed in E.coli and Tn-5B1-4 cells, respectively . The recombinant fusion protein has a subunit molecular mass of 68 kD and is recognized by both anti-CuZn-SOD and anti-tetanustoxin antibody . By pyrogallol autooxidation assay, it is shown that the CuZn-SOD moiety retains substantial enzymatic activity, where the TTC moiety might deliver the fusion protein to neurons in central nervous system . So, CuZn-SOD/TTC may be a useful agent for the targeted delivery of CuZn-SOD to neurons.

Bioinformatics, 2002 May, 18(5), 661 - 5
PGAAS: a prokaryotic genome assembly assistant system; Yu Z et al.; MOTIVATION: In order to accelerate the finishing phase of genome assembly, especially for the whole genome shotgun approach of prokaryotic species, we have developed a software package designated prokaryotic genome assembly assistant system (PGAAS) . The approach upon which PGAAS is based is to confirm the order of contigs and fill gaps between contigs through peptide links obtained by searching each contig end with BLASTX against protein databases . RESULTS: We used the contig dataset of the cyanobacterium Synechococcus sp . strain PCC7002 (PCC7002), which was sequenced with six-fold coverage and assembled using the Phrap package . The subject database is the protein database of the cyanobacterium, Synechocystis sp . strain PCC6803 (PCC6803) . We found more than 100 non-redundant peptide segments which can link at least 2 contigs . We tested one pair of linked contigs by sequencing and obtained satisfactory result . PGAAS provides a graphic user interface to show the bridge peptides and pier contigs . We integrated Primer3 into our package to design PCR primers at the adjacent ends of the pier contigs . AVAILABILITY: We tested PGAAS on a Linux (Redhat 6.2) PC machine . It is developed with free software (MySQL, PHP and Apache) . The whole package is distributed freely and can be downloaded as UNIX compress file: ftp://ftp.cbi.pku.edu.cn/pub/software/unix/pgaas1.0.tar.gz . The package is being continually updated.

Trends Plant Sci, 2002 Jun, 7(6), 257 - 63
Peptide transport in plants; Stacey G et al.; Recent completion of the Arabidopsis genome revealed that this organism has ten times more peptide transporters than any other sequenced organism (prokaryote or eukaryote) . These transporters are found in three protein families: the ABC-type transporters; the di- and tripeptide transporters; and the newly described tetra- and pentapeptide oligopetide transporters . The abundance of these transporters suggests that they play diverse and important roles in plant growth and development . Possible substrates for these transporters include glutathione, gamma-glutamyl peptides, hormone-amino acid conjugates, phytosulfokine, peptide-like compounds and peptide phytotoxins . However, the exact role of peptide transport in plants is still undefined.

Genome Biol . 2002;3(5):research0024 . Epub 2002 Apr 26.
Evolution of gene fusions: horizontal transfer versus independent events; Yanai I et al.; BACKGROUND: Gene fusions can be used as tools for functional prediction and also as evolutionary markers . Fused genes often show a scattered phyletic distribution, which suggests a role for processes other than vertical inheritance in their evolution . RESULTS: The evolutionary history of gene fusions was studied by phylogenetic analysis of the domains in the fused proteins and the orthologous domains that form stand-alone proteins . Clustering of fusion components from phylogenetically distant species was construed as evidence of dissemination of the fused genes by horizontal transfer . Of the 51 examined gene fusions that are represented in at least two of the three primary kingdoms (Bacteria, Archaea and Eukaryota), 31 were most probably disseminated by cross-kingdom horizontal gene transfer, whereas 14 appeared to have evolved independently in different kingdoms and two were probably inherited from the common ancestor of modern life forms . On many occasions, the evolutionary scenario also involves one or more secondary fissions of the fusion gene . For approximately half of the fusions, stand-alone forms of the fusion components are encoded by juxtaposed genes, which are known or predicted to belong to the same operon in some of the prokaryotic genomes . This indicates that evolution of gene fusions often, if not always, involves an intermediate stage, during which the future fusion components exist as juxtaposed and co-regulated, but still distinct, genes within operons . CONCLUSION: These findings suggest a major role for horizontal transfer of gene fusions in the evolution of protein-domain architectures, but also indicate that independent fusions of the same pair of domains in distant species is not uncommon, which suggests positive selection for the multidomain architectures.

Proc Natl Acad Sci U S A, 1981 Nov, 78(11), 6744 - 8
The bacteriorhodopsin gene; Dunn R et al.; The bacteriorhodopsin gene has been identified in a 5.3-kilobase restriction endonuclease fragment isolated from Halobacterium halobium DNA, using a cloned cDNA fragment as the probe . Of the 1229 nucleotides whose sequence was determined in the genomic fragment, 786 correspond to the structural gene of bacteriorhodopsin, 360 are upstream from the initiator methionine codon, and 83 are downstream from the COOH terminus . The bacteriorhodopsin gene codes for a precursor sequence of 13 amino acids at the NH2 terminus, 248 amino acids that are present in the mature protein and an additional aspartic acid at the COOH terminus . This determination of the DNA sequence for an archaebacterial gene reveals that the standard genetic code is used; however, there is a marked preference for either G or C in the third codon position . The gene does not contain any intervening sequences and no prokaryotic promoter can be identified in the region immediately upstream from the structural gene . The bacteriorhodopsin mRNA contains at the 5' terminus only three nucleotides beyond the initiating AUG codon and this terminus can form a hairpin structure . Immediately downstream from this structure there is a sequence complementary to the 3' terminus of H . halobium 16S rRNA.

Trends Genet, 2002 May, 18(5), 228 - 32
Purifying and directional selection in overlapping prokaryotic genes; Rogozin IB et al.; In overlapping genes, the same DNA sequence codes for two proteins using different reading frames . Analysis of overlapping genes can help in understanding the mode of evolution of a coding region from noncoding DNA . We identified 71 pairs of convergent genes, with overlapping 3' ends longer than 15 nucleotides, that are conserved in at least two prokaryotic genomes . Among the overlap regions, we observed a statistically significant bias towards the 123:132 phase (i.e . the second codon base in one gene facing the degenerate third position in the second gene) . This phase ensures the least mutual constraint on nonconservative amino acid replacements in both overlapping coding sequences . The excess of this phase is compatible with directional (positive) selection acting on the overlapping coding regions . This could be a general evolutionary mode for genes emerging from noncoding sequences, in which the protein sequence has not been subject to selection.

Trends Genet, 2002 May, 18(5), 223 - 6
Antisense RNAs everywhere?
Wagner EG, Flardh K.
In recent years, systematic searches of both prokaryote and eukaryote genomes have identified a staggering number of small RNAs, the biological functions of which remain unknown . Small RNA-based regulators are well known from bacterial plasmids . They act on target RNAs by sequence complementarity; that is, they are antisense RNAs . Recent findings suggest that many of the novel orphan RNAs encoded by bacterial and eukaryotic chromosomes might also belong to a ubiquitous, heterogeneous class of antisense regulators of gene expression.

Eur Biophys J, 2002 Mar, 31(1), 14 - 25
Mechanosensitive channels of bacteria and archaea share a common ancestral origin; Kloda A et al.; The ubiquity of mechanosensitive (MS) ion channels set off a search for their functional homologues in archaea, the third domain of life . A new MS channel was identified in the archaeon Methanococcus jannaschii by using the TM1 transmembrane domain of the bacterial MS channel of large conductance, MscL, as a genetic probe to search the archaeal genomic database for MS channel homologues . The hypothetical protein MJ0170 (MscMJ) was found to harbor two MscL-like TM1 structural motifs and showed a high degree of se quence and secondary structure conservation with MscS (YggB) homologues . The alignment of sequences of MscL, MscS and MscMJ homologues further revealed that bacterial and archaeal channels form a phylogenetic tree composed of three main branches and share a common ancestral origin . This suggests the evolution of prokaryotic MS channels via gene duplication of a MscL-like progenitor gene followed by divergence, fur ther indicating that the common ancestor of the prokaryotic MS channels most likely resembled MscL . When expressed in E . coli and functionally examined by the patch clamp, the MscMJ protein behaved as a MS channel with a conductance of 270 pS in 200 mM KCl and a cation selectivity (PK/PC}) of approximately 6 . The structural and functional homologue of MscMJ, MscMJLR, was identified as a second type of MS channel in M . jannaschii . The channel has a conductance of approximately 2 nS, rectifies with voltage and shares cation selectivity with MscMJ . The stoichiometry of both types of MS channels revealed that the free energy of activation, deltaG0 approximately 7kT, obtained for MscMJ matches the one calculated for MscS, deltaG0 approximately 5kT, whereas the free energy of activation approximately deltaG0 approximately 18kT of MscMJLR resembles more the deltaG0 = 14-19kT reported for MscL . The presence of two types of MS channels discovered in the cell envelope of M . jannaschii indicates that multiplicity of MS channels in prokaryotes is a necessary element for their survival in the habitats frequently challenged by sudden changes in osmolarity . Further functional and phylogenetic study of MS channels from all three domains of the universal phylogenetic tree may help to understand the evolution and common biophysical principles that govern mechanosensory transduction.

Genome Res, 2002 Jun, 12(6), 916 - 29
Comparison of the small molecule metabolic enzymes of Escherichia coli and Saccharomyces cerevisiae; Jardine O et al.; The comparison of the small molecule metabolism pathways in Escherichia coli and Saccharomyces cerevisiae (yeast) shows that 271 enzymes are common to both organisms . These common enzymes involve 384 gene products in E . coli and 390 in yeast, which are between one half and two thirds of the gene products of small molecule metabolism in E . coli and yeast, respectively . The arrangement and family membership of the domains that form all or part of 374 E . coli sequences and 343 yeast sequences was determined . Of these, 70% consist entirely of homologous domains, and 20% have homologous domains linked to other domains that are unique to E . coli, yeast, or both . Over two thirds of the enzymes common to the two organisms have sequence identities between 30% and 50% . The remaining groups include 13 clear cases of nonorthologous displacement . Our calculations show that at most one half to two thirds of the gene products involved in small molecule metabolism are common to E . coli and yeast . We have shown that the common core of 271 enzymes has been largely conserved since the separation of prokaryotes and eukaryotes, including modifications for regulatory purposes, such as gene fusion and changes in the number of isozymes in one of the two organisms . Only one fifth of the common enzymes have nonhomologous domains between the two organisms . Around the common core very different extensions have been made to small molecule metabolism in the two organisms.

Annu Rev Biochem, 2002, 71, 17 - 50 Epub 2001 Nov 09.
Error-prone repair DNA polymerases in prokaryotes and eukaryotes; Goodman MF; DNA repair is crucial to the well-being of all organisms from unicellular life forms to humans . A rich tapestry of mechanistic studies on DNA repair has emerged thanks to the recent discovery of Y-family DNA polymerases . Many Y-family members carry out aberrant DNA synthesis-poor replication accuracy, the favored formation of non-Watson-Crick base pairs, efficient mismatch extension, and most importantly, an ability to replicate through DNA damage . This review is devoted primarily to a discussion of Y-family polymerase members that exhibit error-prone behavior . Roles for these remarkable enzymes occur in widely disparate DNA repair pathways, such as UV-induced mutagenesis, adaptive mutation, avoidance of skin cancer, and induction of somatic cell hypermutation of immunoglobulin genes . Individual polymerases engaged in multiple repair pathways pose challenging questions about their roles in targeting and trafficking . Macromolecular assemblies of replication-repair "factories" could enable a cell to handle the complex logistics governing the rapid migration and exchange of polymerases.

Biochemistry, 2002 Jun 11, 41(23), 7490 - 500
Functional and structural characterization of a synthetic peptide representing the N-terminal domain of prokaryotic pyruvate dehydrogenase; Hengeveld AF et al.; A synthetic peptide (Nterm-E1p) is used to characterize the structure and function of the N-terminal region (amino acid residues 4-45) of the pyruvate dehydrogenase component (E1p) from the pyruvate dehydrogenase multienzyme complex (PDHC) from Azotobacter vinelandii . Activity and binding studies established that Nterm-E1p specifically competes with E1p for binding to the dihydrolipoyl transacetylase component (E2p) of PDHC . Moreover, the experiments show that the N-terminal region of E1p forms an independent folding domain that functions as a binding domain . CD measurements, two-dimensional (2D) (1)H NMR analysis, and secondary structure prediction all indicate that Nterm-E1p has a high alpha-helical content . Here a structural model of the N-terminal domain is proposed . The peptide is present in two conformations, the population of which depends on the sample conditions . The conformations are designated "unfolded" at pH > or =6 and "folded" at pH <5 . The 2D (1)H TOCSY spectrum of a mixture of folded and unfolded Nterm-E1p shows exchange cross-peaks that "link" the folded and unfolded state of Nterm-E1p . The rate of exchange between the two species is in the range of 0.5-5 s(-1) . Sharp resonances in the NMR spectra of wild-type E1p demonstrate that this 200 kDa enzyme contains highly flexible regions . The observed dynamic character of E1p and of Nterm-E1p is likely required for the binding of the E1p dimer to the two different binding sites on E2p . Moreover, the flexibility might be essential in sustaining the allosteric properties of the enzyme bound in the complex.

J Chemother, 1991 Jan, 3 Suppl 1, 24 - 7
Macrolides: present and future . An appraisal of in-vitro activity and pharmacokinetic behavior; Furneri PM et al.; Macrolides belong to a large family of drugs called macrocyclic antibiotics, that can be divided into four groups: macrolactamas or ansamycins, polyene macrolides, macrolide-like compounds and macrolide antibiotics . Macrolide antibiotics comprise a large group of drugs, which are very much alike, characterized by large lactonic cycle with 12, 14, 15 or 16 atoms, to which are bound sugar and/or aminosugar . Although there are very large structural differences, macrolides represent a homogeneous group of drugs for which both a spectrum and a mode of action are similar . Macrolide antibiotics bind to the large subunit of prokaryotic ribosomes and perturb protein synthesis . The antimicrobial activity of macrolides is broad spectrum, being exhibited against gram-positive and gram-negative bacteria, including actinomyces and mycobacteria, as well as against treponemes, mycoplasmas, chlamydiae, rickettsias and also against some protozoa . Depending on the drug concentrations, bacterial species and phase of growth, macrolides may be bacteriostatic or bactericidal . Macrolides show high tissue/serum rations and greater concentrations of antibiotics are though to be achieved at the presumed site of infection . Such findings are in agreement with the apparent volumes of distribution for all the macrolide drugs, which are generally large . The worldwide resurgence of interest in macrolide antibiotics has yielded several semisynthetic derivatives of 14- and 16-membered macrolides which have progressed to clinical trials.

Science, 2002 May 31, 296(5573), 1627 - 30
Specialized DNA polymerases, cellular survival, and the genesis of mutations; Friedberg EC et al.; Cell death caused by arrested replication of damaged or structurally altered DNA can be avoided in prokaryotic and eukaryotic cells by multiple DNA polymerases that are specialized to bypass DNA damage . Some of these polymerases perform such translesion DNA synthesis of specific types of damage with high genetic fidelity . However, they exhibit greatly reduced fidelity when they operate on undamaged DNA or on DNA with lesions that are (apparently) not cognate substrates . The low fidelity of some of these specialized polymerases when copying undamaged DNA may be physiologically functional, including generating immunoglobulin diversity.

Appl Environ Microbiol, 2002 Jun, 68(6), 2633 - 6
Characterization of the oxygen tolerance of a hydrogenase linked to a carbon monoxide oxidation pathway in Rubrivivax gelatinosus; Maness PC et al.; A hydrogenase linked to the carbon monoxide oxidation pathway in Rubrivivax gelatinosus displays tolerance to O2 . When either whole-cell or membrane-free partially purified hydrogenase was stirred in full air (21% O2, 79% N2), its H2 evolution activity exhibited a half-life of 20 or 6 h, respectively, as determined by an anaerobic assay using reduced methyl viologen . When the partially purified hydrogenase was stirred in an atmosphere containing either 3.3 or 13% O2 for 15 min and evaluated by a hydrogen-deuterium (H-D) exchange assay, nearly 80 or 60% of its isotopic exchange rate was retained, respectively . When this enzyme suspension was subsequently returned to an anaerobic atmosphere, more than 90% of the H-D exchange activity was recovered, reflecting the reversibility of this hydrogenase toward O2 inactivation . Like most hydrogenases, the CO-linked hydrogenase was extremely sensitive to CO, with 50% inhibition occurring at 3.9 microM dissolved CO . Hydrogen production from the CO-linked hydrogenase was detected when ferredoxins of a prokaryotic source were the immediate electron mediator, provided they were photoreduced by spinach thylakoid membranes containing active water-splitting activity . Based on its appreciable tolerance to O2, potential applications of this hydrogenase are discussed.

Gene, 2002 Feb 20, 285(1-2), 39 - 47
Highly conserved mouse and human brain sulfotransferases: molecular cloning, expression, and functional characterization; Sakakibara Y et al.; By employing reverse transcription-polymerase chain reaction (RT-PCR) in conjunction with 5'-rapid amplification of cDNA ends technique, we have cloned a novel mouse sulfotransferase cDNA . Database search led to the identification of a human gene encoding the homologue of this newly discovered mouse sulfotransferase . RT-PCR technique was employed to clone the cDNA encoding the human enzyme . Sequence analysis revealed that the novel mouse and human sulfotransferases display nearly 98% identity in their amino acid sequences . Their amino acid sequence identity to other known cytosolic sulfotransferases, however, was found to be below 36% . These two highly conserved sulfotransferases therefore appear to belong to a family different from the two major mammalian cytosolic sulfotransferase gene families . Northern blot analysis revealed the neuronal tissue-specific expression of these two novel sulfotransferases . Recombinant mouse and human brain sulfotransferases, expressed using the pGEX-2TK prokaryotic expression system and purified from transformed Escherichia coli cells, migrated as 33 kD proteins upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Purified mouse and human brain sulfotransferases displayed enzymatic activities toward endogenous and xenobiotic compounds, including L-triiodothyronine, thyroxine, estrone, p-nitrophenol, 2-naphthylamine, and 2-naphthol . Using mouse brain filtrate as substrate, both brain sulfotransferases were shown to catalyze specifically the sulfation of only a few compounds.

Biochem J, 2002 Oct 1, 367(Pt 1), 287 - 93
Isolation and structural elucidation of biologically active phospholipids from Scytonema julianum (cyanobacteria); Antonopoulou S et al.; The role of platelet-activating factor (PAF) as a mediator appeared in rather primitive organisms like protozoans and was maintained in more evolved organisms . No reports exist for the presence of PAF or PAF analogues - or even compounds that exhibit PAF-like activity - in cyanobacteria, even though they belong to a a group of organisms at a low evolutionary level where the content of alkylacyl forms of ether lipids is expected to be high . In addition, cyanobacteria serve as a rich source of novel bioactive metabolites . In the present study the total lipids of a strain of Scytonema julianum, a filamentous cyanobacterium isolated from a Greek cave, were separated into neutral lipids and phospholipids, the latter being further fractionated by HPLC . Each phospholipid fraction was tested in vitro for its ability to inhibit PAF-, arachidonic acid- and ADP-induced washed-rabbit-platelet aggregation and/or to cause platelet aggregation . Two types of phospholipids causing platelet aggregation were detected and shown to be an acetylsphingomyelin and an acylacetylglycerol phosphoacetylated glycolipid . The existence of the sphingomyelin analogues is very important, since ceramides, cerebrosides and related lipids are intracellular second messengers . The identification of the phosphoglycoglycerolipid demonstrates a new type of lipid in cyanobacteria, namely one that exhibits a biological activity very similar to that of PAF . Its presence reinforces the concept that PAF is a member of a large family of lipid mediators, apparently having different physiological roles in prokaryotic and eukaryotic organisms . In addition, Scytonema julianum contains a phosphatidylcholine (C(16:0)/(18:2)), even though bacteria in general seldom contain choline-containing phosphoacylglycerols.

Nature, 2002 May 30, 417(6888), 567 - 71
Functional relationship of cytochrome c(6) and plastocyanin in Arabidopsis; Gupta R et al.; Photosynthetic electron carriers are important in converting light energy into chemical energy in green plants . Although protein components in the electron transport chain are largely conserved among plants, algae and prokaryotes, there is thought to be a major difference concerning a soluble protein in the thylakoid lumen . In cyanobacteria and eukaryotic algae, both plastocyanin and cytochrome c(6) mediate electron transfer from cytochrome b(6)f complex to photosystem I . In contrast, only plastocyanin has been found to play the same role in higher plants . It is widely accepted that cytochrome c(6) has been evolutionarily eliminated from higher-plant chloroplasts . Here we report characterization of a cytochrome c(6)-like protein from Arabidopsis (referred to as Atc6) . Atc6 is a functional cytochrome c localized in the thylakoid lumen . Electron transport reconstruction assay showed that Atc6 replaced plastocyanin in the photosynthetic electron transport process . Genetic analysis demonstrated that neither plastocyanin nor Atc6 was absolutely essential for Arabidopsis growth and development . However, plants lacking both plastocyanin and Atc6 did not survive.

Genomics, 2002 Jun, 79(6), 881 - 9
Genomic organization of mouse and human 65 kDa FK506-binding protein genes and evolution of the FKBP multigene family; Patterson CE et al.; FK506-binding proteins (FKBPs) are peptidyl-prolyl cis/trans isomerases PPIases) that bind the immunosuppressive drug FK506 . Of the many eukaryotic FKBPs that have been identified, FKBP65 is an endoplasmic reticulum-localized protein that associates with tropoelastin in the secretory pathway . Unlike any other FKBP characterized so far, FKBP65 is developmentally regulated and may be intimately involved in organogenesis . Here, we report the isolation, sequencing, and genomic organization of the mouse FKBP65 gene (Fkbp10) and provide a comparison with the human ortholog . Mouse Fkbp10 contains 10 exons and 9 introns encompassing 8.5 kb . The exon-intron organization of Fkbp10 displays a pattern of repetition that reflects the coding sequence of the four PPIase, or FK506-binding, domains present in the mature protein . The exon organization of the PPIase domains differs from that of the other FKBP family members . The evolution of the FKBP65 gene and other members of the FKBP multigene family were therefore investigated from a taxonomically diverse array of prokaryotic and eukaryotic taxa . These analyses suggest that the FKBP multigene family emerged early in the evolutionary history of eukaryotes, and during that time some members, including the FKBP65 gene, have experienced gene elongation by means of PPIase domain duplication . (c)2002 Elsevier Science (USA).

Microb Ecol, 2001 Jul, 42(1), 80 - 86
Toxin-Producing Anabaena flos-aquae Induces Settling of Chlamydomonas reinhardtii, a Competing Motile Alga; Kearns KD et al.; Toxin production is an adaptation that allows cyanobacteria in resource-limiting environments to ameliorate the effects of herbivory and competition with other phototrophs . We demonstrate that the cyanobacterial toxins anatoxin-a and microcystin-LR paralyze the motile green alga Chlamydomonas reinhardtii . In addition, both purified toxins and cyanobacterial extracellular products containing these toxins cause the alga to settle faster than in nontoxic media . In microcosm experiments, the presence of either the cyanobacterium or its extracellular products induce settling in the alga, similar to the response observed with the addition of both anatoxin-a and microcystin-LR . The cyanobacterial production of paralyzing toxins represents a novel mechanism for phytoplankton settling . This prokaryotic/eukaryotic chemical interaction may create a competitor-free zone for cyanobacteria in lake environments, predicating optimal conditions for a toxic cyanobacterial bloom.

Microb Ecol, 2001 Jul, 42(1), 1 - 10
Thermodynamic and Kinetic Requirements in Anaerobic Methane Oxidizing Consortia Exclude Hydrogen, Acetate, and Methanol as Possible Electron Shuttles; Sorensen KB et al.; Anaerobic methane oxidation (AMO) has long remained an enigma in microbial ecology . In the process the net reaction appears to be an oxidation of methane with sulfate as electron acceptor . In order to explain experimental data such as effects of inhibitors and isotopic signals in biomarkers it has been suggested that the process is carried out by a consortium of bacteria using an unknown compound to shuttle electrons between the participants . The overall change in free energy during AMO with sulfate is very small (?22 kJ mol-1) at in situ concentrations of methane and sulfate . In order to share the available free energy between the members of the consortium, the concentration of the intermediate electron shuttle compound becomes crucial . Diffusive flux of a substrate (i.e, the electron shuttle) between bacteria requires a stable concentration gradient where the concentration is higher in the producing organism than in the consuming organism . Since changes in concentrations cause changes in reaction free energies, the diffusive flux of a catabolic product/substrate between bacteria is associated with a net loss of available energy . This restricts maximal inter-bacterial distances in consortia composed of stationary bacteria . A simple theoretical model was used to describe the relationship between inter-bacterial distances and the energy lost due to concentration differences in consortia . Key parameters turned out to be the permissible concentration range of the electron shuttle in the consortium (i.e., the concentration range that allows both participants to gain sufficient energy) and the stoichiometry of the partial reactions . The model was applied to two known consortia degrading ethanol and butyrate and to four hypothetical methane-oxidizing consortia (MOC) based on interspecies transfer of hydrogen, methanol, acetate, or formate, respectively . In the first three MOCs the permissible distances between producers and consumers of the transferred compounds were less than two times prokaryotic cell wall diameters . Consequently, it is not possible that a MOC can be based on inter-species transfer of hydrogen, methanol, or acetate . Formate, on the other hand, is a possible shuttle candidate provided the bacteria are attached to one another . In general the model predicts that members of consortia thriving on low energy such as the MOC must adhere to each other and utilize a compound for the exchange of electrons that has a high permissible concentration range and a high diffusion coefficient and transfers as many electrons as possible per molecule.

Protein Eng, 2002 May, 15(5), 413 - 8
Enhanced proliferative effects of a baculovirus-produced fusion protein of insulin-like growth factor and alpha(1)-proteinase inhibitor and improved anti-elastase activity of the inhibitor with glutamate at position 351; Sandoval C et al.; Alpha(1)-proteinase inhibitor (API) was coupled at the C-terminus of a human insulin-like growth factor (IGF) analog to facilitate its production in insect cells . This fusion protein significantly increased thymidine incorporation into HL-60 cells as compared with the incorporation observed with an equivalent molar mixture of the IGF analog and API . The M351E variant of API has been previously shown to reduce aggregate formation in prokaryotic expression systems . When the oxidation-sensitive methionine 351 of the inhibitor was changed to glutamate, the M351E variant was secreted in larger amounts from insect cells than the corresponding fusion protein with wild-type API . The M351E fusion protein and the corresponding chimera containing the wild-type API were tested for their capacity to inhibit human neutrophil elastase . The M351E variant was a more potent elastase inhibitor than the fusion protein containing the wild-type analog, whereas the proliferative activity of both chimeras was identical . The described mitogenic effect of the chimera and the improved anti-elastase activity of the M351E variant are two ideal properties for therapeutic agents acting in pathological situations where cell proliferation and inhibition of neutrophil elastase have to take place simultaneously, such as during wound healing.

Nucleic Acids Res, 2002 Jun 1, 30(11), 2478 - 83
Fast algorithms for large-scale genome alignment and comparison; Delcher AL et al.; We describe a suffix-tree algorithm that can align the entire genome sequences of eukaryotic and prokaryotic organisms with minimal use of computer time and memory . The new system, MUMmer 2, runs three times faster while using one-third as much memory as the original MUMmer system . It has been used successfully to align the entire human and mouse genomes to each other, and to align numerous smaller eukaryotic and prokaryotic genomes . A new module permits the alignment of multiple DNA sequence fragments, which has proven valuable in the comparison of incomplete genome sequences . We also describe a method to align more distantly related genomes by detecting protein sequence homology . This extension to MUMmer aligns two genomes after translating the sequence in all six reading frames, extracts all matching protein sequences and then clusters together matches . This method has been applied to both incomplete and complete genome sequences in order to detect regions of conserved synteny, in which multiple proteins from one organism are found in the same order and orientation in another . The system code is being made freely available by the authors.

EMBO Rep, 2002 Jun, 3(6), 557 - 62 Epub 2002 May 24.
A Toc75-like protein import channel is abundant in chloroplasts; Eckart K et al.; Chloroplasts import post-translationally most of their constituent polypeptides via two distinct translocon units located in the outer and inner envelope . The protein import channel of the translocon of the outer envelope of chloroplasts, Toc75, is the most abundant protein in that membrane . We identify a novel Toc75 homologous protein, atToc75-V, a prominent protein that is clearly localized in the chloroplastic outer envelope . Phylogenetic analysis indicates that Toc75-V is more closely related to its prokaryotic ancestors than to Toc75 from plants . The presence of a second translocation channel suggests that alternative, previously unrecognized import routes into chloroplasts might exist.

Biochemistry, 2002 Jun 4, 41(22), 7108 - 15
Functional role of the prokaryotic proline-tRNA synthetase insertion domain in amino acid editing; Wong FC et al.; Aminoacyl-tRNA synthetases catalyze the attachment of specific amino acids to cognate tRNAs in a two-step process that is critical for the faithful translation of genetic information . During the first chemical step of tRNA aminoacylation, noncognate amino acids that are smaller than or isosteric with the cognate substrate can be misactivated . Thus, to maintain high accuracy during protein translation, some synthetases have evolved an editing mechanism . Previously, we showed that class II Escherichia coli proline-tRNA synthetase (ProRS) is capable of (1) weakly misactivating Ala, (2) hydrolyzing the misactivated Ala-AMP in a reaction known as pretransfer editing, and (3) deacylating a mischarged Ala-tRNA(Pro) variant via a post-transfer editing pathway . In contrast to most systems where an editing function has been established, pretransfer editing by E . coli ProRS occurs in a tRNA-independent fashion . However, neither the pre- nor the post-transfer editing active site(s) has been identified . Sequence analyses revealed that most prokaryotic ProRSs possess a large insertion domain (INS) between class II conserved motifs 2 and 3 . The function of the approximately 180-amino acid INS in E . coli ProRS is the subject of this investigation . Alignment-guided Ala scanning mutagenesis was carried out to test conserved amino acid residues present in the INS for their role in pre- and post-transfer editing . Our biochemical data and modeling studies suggest that the prokaryotic INS plays a critical role in editing and that this activity resides in a domain that is functionally and structurally distinct from the aminoacylation active site.

Biosci Rep, 1983 Jun, 3(6), 527 - 38
Multienzyme complexes of eukaryotic aminoacyl-tRNA synthetases; Dang CV et al.; Eukaryotic aminoacyl-tRNA synthetases, unlike their prokaryotic counterparts, may occur as high-Mr multienzyme complexes . Recently, successful purification of synthetase complexes makes possible the elucidation of the structural organization of these high-Mr complexes . Although their physiological significance remains unknown, recent studies suggest some possible functional roles for these complexes.

Proc Natl Acad Sci U S A, 2002 May 28, 99(11), 7658 - 62
Benthic eukaryotic diversity in the Guaymas Basin hydrothermal vent environment; Edgcomb VP et al.; Molecular microbial ecology studies have revealed remarkable prokaryotic diversity in extreme hydrothermal marine environments . There are no comparable reports of culture-independent surveys of eukaryotic life in warm, anoxic marine sediments . By using sequence comparisons of PCR-amplified small subunit ribosomal RNAs, we characterized eukaryotic diversity in hydrothermal vent environments of Guaymas Basin in the Gulf of California . Many sequences from these anoxic sediments and the overlaying seawater represent previously uncharacterized protists, including early branching eukaryotic lineages or extended diversity within described taxa . At least two mechanisms, with overlapping consequences, account for the eukaryotic community structure of this environment . The adaptation to anoxic environments is evidenced by specific affinity of environmental sequences to aerotolerant anaerobic species in molecular trees . This pattern is superimposed against a background of widely distributed aerophilic and aerotolerant protists, some of which may migrate into and survive in the sediment whereas others (e.g., phototrophs) are simply deposited by sedimentary processes . In contrast, bacterial populations in these sediments are primarily characteristic of anoxic, reduced, hydrocarbon-rich sedimentary habitats.

Chem Biol, 2002 May, 9(5), 639 - 45
Crystal structure of a squalene cyclase in complex with the potential anticholesteremic drug Ro48-8071; Lenhart A et al.; Squalene-hopene cyclase (SHC) catalyzes the conversion of squalene into pentacyclic compounds . It is the prokaryotic counterpart of the eukaryotic oxidosqualene cyclase (OSC) that catalyzes the steroid scaffold formation . Because of clear sequence homology, SHC can serve as a model for OSC, which is an attractive target for anticholesteremic drugs . We have established the crystal structure of SHC complexed with Ro48-8071, a potent inhibitor of OSC and therefore of cholesterol biosynthesis . Ro48-8071 is bound in the active-center cavity of SHC and extends into the channel that connects the cavity with the membrane . The binding site of Ro48-8071 is largely identical with the expected site of squalene; it differs from a previous model based on photoaffinity labeling . The knowledge of the inhibitor binding mode in SHC is likely to help develop more potent inhibitors for OSC.

Planta, 2002 Jun, 215(2), 167 - 76 Epub 2002 Feb 26.
Overexpression of the Arabidopsis thaliana MinE1 bacterial division inhibitor homologue gene alters chloroplast size and morphology in transgenic Arabidopsis and tobacco plants; Reddy MS et al.; Higher-plant chloroplast division requires some of the same genes that are involved in prokaryotic cell division . These include the FtsZ and MinD proteins . Other genes that might be involved in higher-plant chloroplast division have yet to be characterized . The Arabidopsis thaliana (L.) Heynh . MinE ( AtMinE1) gene was identified in the genomic database, isolated by reverse transcription-polymerase chain reaction and constitutively expressed in tobacco ( Nicotiana tabacum L.) and Arabidopsis plants in both the sense and antisense orientation . Confocal and electron-microscopic analysis of the sense-overexpressing AtMinE1 transgenic tobacco and Arabidopsis plants revealed that the chloroplasts were abnormal in size and shape compared to wild-type Arabidopsis and tobacco chloroplasts . Our results, based on the overexpression of the AtMinE1 gene in tobacco and Arabidopsis, confirm that the AtMinE1 gene is involved in plant chloroplast division.

Arch Microbiol, 2002 Jun, 177(6), 441 - 50 Epub 2002 Mar 21.
Sequence and phylogenetic analyses of the twin-arginine targeting (Tat) protein export system; Yen MR et al.; Twin-arginine targeting (Tat) protein secretion systems consist of two protein types, members of the TatA and TatC families . Homologues of these proteins are found in many archaea, bacteria, chloroplasts and mitochondria . Every prokaryotic organism with a fully sequenced genome exhibits either neither family member, or between one and three paralogues of these two family members . The Arabidopsis thaliana genome encodes three of each . Although many mitochondrially encoded TatC homologues have been identified, corresponding TatA homologues have not been found in this organelle . Phylogenetic analyses reveal that most prokaryotic Tat systems consist of one TatC homologue and two sequence-divergent TatA homologues (TatA and TatB) . When only one TatA homologue is present, TatB is missing, and when three TatA homologues are present, the third one arose by duplication of TatA, not TatB . Further, homologues most resembling TatB are more sequence-divergent than those more closely resembling TatA . In contrast to the TatA family, the TatC family shows phylogenetic clustering in strict accordance with organismal type . These results are discussed in terms of their probable structural, functional and evolutionary significance.

Pigment Cell Res, 2002 Jun, 15(3), 162 - 73
Molecular anatomy of tyrosinase and its related proteins: beyond the histidine-bound metal catalytic center; Garcia-Borron JC et al.; The structure of tyrosinase (Tyr) is reviewed from a double point of view . On the one hand, by comparison of all Tyr found throughout nature, from prokaryotic organisms to mammals and on the other, by comparison with the tyrosinase related proteins (Tyrps) that appeared late in evolution, and are only found in higher animals . Their structures are reviewed as a whole rather than focused on the histidine (His)-bound metal active site, which is the part of the molecule common to all these proteins . The availability of crystallographic data of hemocyanins and recently of sweet potato catechol oxidase has improved the model of the three-dimensional structure of the Tyr family . Accordingly, Tyr has a higher structural disorder than hemocyanins, particularly at the CuA site . The active site seems to be characterized by the formation of a hydrophobic pocket with a number of conserved aromatic residues sited close to the well-known His . Other regions specific of the mammalian enzymes, such as the cytosolic C-terminal tail, the cysteine clusters, and the N-glycosylation sequons, are also discussed . The complete understanding of the Tyr copper-binding domain and the characterization of the residues determinant of the relative substrate affinities of the Tyrps will improve the design of targeted mutagenesis experiments to understand the different catalytic capabilities of Tyr and Tyrps . This may assist future aims, from the design of more efficient bacterial Tyr for biotechnological applications to the design of inhibitors of undesirable fruit browning in vegetables or of color skin modulators in animals.

Eur J Biochem, 2002 May, 269(10), 2601 - 6
Histidine mutagenesis of Arabidopsis thaliana pyruvate dehydrogenase kinase; Tovar-Mendez A et al.; Pyruvate dehydrogenase kinase (PDK) is the primary regulator of flux through the mitochondrial pyruvate dehydrogenase complex (PDC) . Analysis of the primary amino-acid sequences of PDK from various sources reveals that these enzymes include the five domains characteristic of prokaryotic two-component His-kinases, despite the fact that PDK exclusively phosphorylates Ser residues in the E1alpha subunit of the PDC . This seeming contradiction might be resolved if the PDK-catalyzed reaction employed a phospho-His intermediate . The results from pH-stability studies of autophosphorylated Arabidopsis thaliana PDK did not provide any support for a phospho-His intermediate . Furthermore, site-directed mutagenesis of the two most likely phosphotransfer His residues (H121 and H168) did not abolish either PDK autophosphorylation or the ability to transphosphorylate E1alpha . Thus, PDK is a unique type of protein kinase having a His-kinase-like sequence but Ser-kinase activity.

Microb Ecol, 2001 Aug, 42(2), 109 - 115
Group-Specific PCR Primers to Amplify 24S a-Subunit rRNA Genes from Kinetoplastida (Protozoa) Used in Denaturing Gradient Gel Electrophoresis; Rasmussen LD et al.; We developed and tested a set of primers for amplification of a region of the 24S a-subunit rRNA genes (24S rDNA) specific to Kinetoplastida (Protozoa) . The reverse primer was supplied with a GC rich region in the 5? end in order to make the PCR product suitable for analysis by denaturing gradient gel electrophoresis (DGGE) . PCR product was obtained from all the kinetoplastids tested and no PCR product was obtained from any other Eukaryotes or Prokaryotes tested . It was possible to distinguish between all pure cultures of kinetoplastids by denaturing gradient gel electrophoresis in gels ranging from 20% to 60% denaturants . PCR-DGGE analysis of DNA purified from lake sediment revealed approximately 20 bands indicating high kinetoplastid diversity . Direct cloning and sequencing of 24S rDNA sequences retrieved from the lake sediment by PCR also showed high kinetoplastid diversity . Of 43 clones, 27 different sequences were found . Alignments and phylogenetic analysis showed that a majority of the sequences were most closely related to the Bodonidae . Four sequences were closer to the Trypanosomatidae, whereas three sequences fell outside both groups . The PCR-DGGE procedure developed in this study has been shown to be useful for distinguishing between different kinetoplastid species . Thus, it may be a useful tool for evaluating the genetic diversity of this group in environmental samples, e.g., as a result of perturbation . Another possible application of this method is in fast and accurate screening for the presence and identification of pathological parasitic Kinetoplastida from environmental samples and for diagnostics of human and animal infections.

Microb Ecol, 2001 Oct, 42(3), 338 - 349
N2-Fixation in Cyanobacterial Mats from Ponds on the McMurdo Ice Shelf, Antarctica; Fernandez-Valiente E et al.; We have investigated the ecological importance of N2-fixation in cyanobacterial mats, dominated by oscillatorean species, in ponds of the Bratina Island area of the McMurdo Ice Shelf, Antarctica (78 degrees S, 166 degrees E) . Nitrogenase activity, estimated as acetylene reducing activity (ARA), was found in all the mats investigated (n = 16) . The average ARA was 75.9 mmol ethylene m-2 h-1, ranging from 6 to 201 mmol ethylene m-2 h-1 . Nitrogenase activity was positively correlated with dissolved reactive phosphorus concentration in pondwater and the C/N ratio of the mat, and was negatively correlated with pondwater NH4+-N concentrations and natural abundance of 15N in the mats . ARA was restricted to the upper, oxic layer of the mats . Experiments conducted to ascribe ARA to different groups of prokaryotes suggested that ARA was mainly conducted by heterocystous cyanobacteria, since no activity was found in the dark and the activity was inhibited by the photosystem II inhibitor DCMU (3-{3,4-dichlorophenyl}-1,1-dimethyl urea) . In spite of 24 h of daylight, nitrogenase activity showed a diel cycle with maximum activity at midday (10-18 h) and minimal activity at early morning (6-10 h) when pond temperatures were at their minima . Light dependency of nitrogenase activity for three cyanobacterial communities showed that the irradiance required for saturating ARA was low, in every case lower than 100 mmol photon m-2s-1 . Irradiance rarely fell below 100 mmol photon m-2s-1 during Antarctic summer days and ARA was likely to be light saturated for much of the time . We estimate that N2 fixation represented on average a N input into the ponds of over 1 g m-2y-1 . This value appears to be the highest N input to this Antarctic ecosystem.

Microb Ecol, 2001 Oct, 42(3), 317 - 327
Use of Metabolic Inhibitors to Characterize Ecological Interactions in an Estuarine Microbial Food Web; DeLorenzo ME et al.; Understanding microbial food web dynamics is complicated by the multitude of competitive or interdependent trophic interactions involved in material and energy flow . Metabolic inhibitors can be used to gain information on the relative importance of trophic pathways by uncoupling selected microbial components and examining the net effect on ecosystem structure and function . A eukaryotic growth inhibitor (cycloheximide), a prokaryotic growth inhibitor (antibiotic mixture), and an inhibitor of photosynthesis (DCMU) were used to examine the trophodynamics of microbial communities from the tidal creek in North Inlet, a salt marsh estuary near Georgetown, South Carolina . Natural microbial communities were collected in the spring, summer, and fall after colonization onto polyurethane foam substrates deployed in the tidal creek . Bacterial abundance and productivity, heterotrophic ciliate and flagellate abundance, and phototrophic productivity, biomass, and biovolume were measured at five time points after inhibitor additions . The trophic responses of the estuarine microbial food web to metabolic inhibitors varied with season . In the summer, a close interdependency among phototrophs, bacteria, and protozoa was indicated, and the important influence of microzooplanktonic nutrient recycling was evident (i.e., a positive feedback loop) . In the fall, phototroph and bacteria interactions were competitive rather than interdependent, and grazer nutrient regeneration did not appear to be an important regulatory factor for bacterial or phototrophic activities . The results indicate a seasonal shift in microbial food web structure and function in North Inlet, from a summer community characterized by microbial loop dynamics to a more linear trophic system in the fall . This study stresses the important role of microbial loops in driving primary and secondary production in estuaries such as North Inlet that are tidally dominated by fluctuations in nutrient supply and a summer phytoplankton bloom.

Microb Ecol, 2001 Oct, 42(3), 306 - 316
Genome Evolution of the Cyanobacterium Nostoc linckia under Sharp Microclimatic Divergence at "Evolution Canyon," Israel; Satish N et al.; We describe the genomic DNA diversity and divergence of the cyanobacterium Nostoc linckia from "Evolution Canyon," a microsite consisting of ecologically contrasting slopes, south-facing slope (SFS) and north-facing slope (NFS), at lower Nahal Oren, Mt . Carmel, Israel . The opposing slopes share their limestone lithology but vary greatly in their ecology, primarily because of different levels of solar radiation (which is six times higher on the SFS than on the NFS) . The warm and xeric SFS displays a tropical African savanna, whereas the cool and mesic NFS displays a temperate South European Mediterranean live-oak maquis shrub forest . The cyanobacterium Nostoc linckia tested here is a sessile microorganism, growing as a carpet on rock surfaces and constantly exposed to environmental fluctuations of solar radiation, temperature, and desiccation . We demonstrate remarkable interslope and intraslope genetic divergence of the genome (including both coding and noncoding regions) of Nostoc linckia, by using 211 AFLP (amplified fragment length polymorphism) DNA molecular marker loci . Genetic polymorphism of N . linckia subpopulations on the ecologically harsher SFS was significantly (p <0.05) higher (p = 99.53%) than was that of the subpopulations on the climatically milder nfs (p = 85.78%) . genetic polymorphism (p) and gene diversity (he) were significantly correlated with variables influencing aridity stress: solar radiation (sr) (rp = 0.956; p = 0.046), temperature (tm) (rp = 0.993; p = 0.0068), and day-night temperature difference (tdd) (rp = 0.975; p = 0.025) . as in other tested organisms from "evolution canyon", but even more exceptionally because of its completely sedentary nature, we suggest that the climatically stressed sfs environment is responsible for this marked increase of genetic polymorphism, which is maintained by the combined evolutionary forces of diversifying and balancing selection . This could highlight the importance of ecological stress and selection in evolution and its remarkable effect on the genetic system across the prokaryotic genome.

FEMS Microbiol Lett, 2002 Apr 23, 210(1), 129 - 33
The iron-containing superoxide dismutase of Ralstonia metallidurans CH34; Roux M et al.; The iron-containing superoxide dismutase (Fe-SOD) of Ralstonia metallidurans CH34 was purified and characterised as a homodimer of 2 x 21500 Da containing one iron atom per monomer and exhibiting all the characteristics of the prokaryotic Fe-SODs except for a higher isoelectric point . The protein was 2-fold overexpressed in the presence of selenite, zinc or paraquat . R . metallidurans CH34 was suggested to contain a gene encoding for a manganese-containing SOD located in the inducible chromate resistance operon . Whatever the culture conditions used in this study, including the presence of chromate, only a Fe-SOD, genetically distinct from the putative Mn-SOD, was detected . This Fe-SOD seems to be the only active superoxide dismutase expressed in R . metallidurans CH34.

FEBS Lett, 2002 May 22, 519(1-3), 191 - 4
Bacterial toxin RelE induces apoptosis in human cells; Yamamoto TA et al.; The bacterial protein RelE severely restricts prokaryotic cell growth, probably by acting as a global inhibitor of translation . It is ubiquitous in prokaryotes as part of the RelE-RelB toxin-antitoxin system, and may be activated by nutritional stress . When the relE gene from Escherichia coli was expressed inducibly in a human osteosarcoma cell line, it was shown to retard growth and to lead to cell death by apoptosis . RelE is therefore unusual among bacterial toxins in possessing broad activity against both prokaryotes and eukaryotes, perhaps by acting on evolutionarily conserved components of the translation machinery.

FEBS Lett, 2002 May 22, 519(1-3), 123 - 7
Identification of a novel prokaryotic HEAT-repeats-containing protein which interacts with a cyanobacterial IscA homolog; Morimoto K et al.; IscA homologs are known to be involved in iron-sulfur cluster formation in various organisms . Recombinant proteins of two IscA homologs from the cyanobacterium Synechocystis PCC 6803, designated SLR1417 and SLR1565, were purified . The absorption spectrum of purified SLR1565 was typical for {2Fe-2S} cluster-containing proteins, whereas that of SLR1417 predominantly showed the presence of the iron ion alone . In the cyanobacterial cell extracts, only SLR1565 was found to form a complex with a novel prokaryotic HEAT-repeats-containing protein, SLR1098 . Thus, the two cyanobacterial IscA protein homologs exist in distinct molecular states, suggesting different cellular roles for these proteins.

Biochemistry, 2002 May 28, 41(21), 6875 - 84
The "second stalk" of Escherichia coli ATP synthase: structure of the isolated dimerization domain; Del Rizzo PA et al.; The b subunit of E . coli F(0)F(1)-ATPase links the peripheral F(1) subunits to the membrane-integral F(0) portion and functions as a "stator", preventing rotation of F(1) . The b subunit is present as a dimer in ATP synthase, and residues 62-122 are required to mediate dimerization . To understand how the b subunit dimer is formed, we have studied the structure of the isolated dimerization domain, b(62-122) . Analytical ultracentrifugation and solution small-angle X-ray scattering (SAXS) indicate that the b(62-122) dimer is extremely elongated, with a frictional ratio of 1.60, a maximal dimension of 95 A, and a radius of gyration of 27 A, values that are consistent with an alpha-helical coiled-coil structure . The crystal structure of b(62-122) has been solved and refined to 1.55 A . The protein crystallized as an isolated, monomeric alpha helix with a length of 90 A . Combining the crystal structure of monomeric b(62-122) with SAXS data from the dimer in solution, we have constructed a model for the b(62-122) dimer in which the two helices form a coiled coil with a right-handed superhelical twist . Analysis of b sequences from E . coli and other prokaryotes indicates conservation of an undecad repeat, which is characteristic of a right-handed coiled coil and consistent with our structural model . Mutation of residue Arg-83, which interrupts the undecad pattern, to alanine markedly stabilized the dimer, as expected for the proposed two-stranded, right-handed coiled-coil structure.

Antimicrob Agents Chemother, 2002 Jun, 46(6), 1805 - 15
Quinolone-binding pocket of DNA gyrase: role of GyrB; Heddle J et al.; DNA gyrase is a prokaryotic type II topoisomerase and a major target of quinolone antibacterials . The majority of mutations conferring resistance to quinolones arise within the quinolone resistance-determining region of GyrA close to the active site (Tyr(122)) where DNA is bound and cleaved . However, some quinolone resistance mutations are known to exist in GyrB . Present structural data suggest that these residues lie a considerable distance from the quinolone resistance-determining region, and it is not obvious how they affect quinolone action . We have made and purified two such mutant proteins, GyrB(Asp(426)-->Asn) and GyrB(Lys(447)-->Glu), and characterized them in vitro . We found that the two proteins behave similarly to GyrA quinolone-resistant proteins . We showed that the mutations exert their effect by decreasing the amount of quinolone bound to a gyrase-DNA complex . We suggest that the GyrB residues form part of a quinolone-binding pocket that includes DNA and the quinolone resistance-determining region in GyrA and that large conformational changes during the catalytic cycle of the enzyme allow these regions to come into close proximity.

IUBMB Life, 2002 Jan, 53(1), 3 - 14
Protein tyrosine phosphatases: dephosphorylating the epidermal growth factor receptor; Tiganis T; Protein tyrosine phosphatases (PTPs) are a large and structurally diverse family of enzymes that are found in eukaryotes, prokaryotes, viruses, and plants . PTPs catalyse the dephosphorylation of tyrosyl phosphorylated proteins and can either antagonise or potentiate protein tyrosine kinase signalling . PTPs regulate fundamental cellular processes and have been implicated in the etiology and pathogenesis of various human diseases . The epidermal growth factor receptor (EGFR) is a widely distributed protein tyrosine kinase that regulates both normal development and plays a role in pathological conditions such as cancer . This review discusses the structure and function of PTPs and focuses on the PTPs that have been implicated in the dephosphorylation of the EGFR and the consequent suppression of EGFR signalling.

Photochem Photobiol, 2002 May, 75(5), 554 - 9
Phytochromes with noncovalently bound chromophores: the ability of apophytochromes to direct tetrapyrrole photoisomerization; Jorissen HJ et al.; Chromophore-apoprotein interactions were studied with recombinant apoproteins, oat phytochrome (phyA) and CphB of the cyanobacterium Calothrix PCC7601, which were both incubated with the bilin compounds biliverdin (BV) IXalpha, phycocyanobilin (PCB) and the 3'-methoxy derivative of PCB . Previously it was shown that CphB and its homolog in Calothrix, CphA, show strong sequence similarities with each other and with the phytochromes of higher and lower plants, despite the fact that CphB carries a leucine instead of a cysteine at the chromophore attachment position and thus holds the chromophore only noncovalently . CphA binds tetrapyrrole chromophores in a covalent, phytochrome-like manner . For both eyanobacterial phytochromes, red and far-red light-induced photochemistry has been reported . Thus, the role of the binding site of CphB in directing the photochemistry of noncovalently bound tetrapyrroles was analyzed in comparison with the apoprotein from phyA phytochrome . Both the aforementioned compounds, which were used as chromophores, are not able to form covalent bonds with a phytochrome-type apoprotein because of their chemical structure (vinyl group at position 3 or methoxy group at position 3') . The BV adducts of both apoproteins showed phytochrome-like photochemistry (formation of red and far-red-absorbing forms of phytochrome {P(r) and P(fr) forms}) . However, incubation of the oat apophytochrome with BV primarily yields a 700 nm form from which the P(r)-P(fr) photochemistry can be initiated and to which the system relaxes in the dark after illumination . The results for CphB were compared with a CphB mutant where the chromophore-binding cysteine had been introduced, which, upon incubation with PCB, shows spectral properties nearly identical with its (covalently binding) CphA homolog . A comparison of the spectral properties (P(r) and P(fr) forms) of all the PCB- and BV-containing chromoproteins reveals that the binding site of the cyanobacterial apoprotein is better suited than the plant (oat) phytochrome to noncovalently incorporate the chromophore and to regulate its photochemistry . Our findings support the proposal that the recently identified phytochrome-like prokaryotic photoreceptors, which do not contain a covalently bound chromophore, may trigger a light-induced physiological response.

Bioinformatics, 2002 Apr, 18(4), 597 - 607
TranScout: prediction of gene expression regulatory proteins from their sequences; Aguilar D et al.; MOTIVATION: The advent of genomics yields thousands of reading frames in search of function . Identification of conserved functional motifs in protein sequences can be helpful for function prediction . RESULTS: A database and a classification of reported DNA-binding protein motifs has been designed . A program ('TranScout') has been developed for the detection and evaluation of conserved motifs in prokaryotic and eukaryotic sequences of proteins with a gene regulatory function . The efficiency of the program is shown in a benchmark against a database obtained from SWISS-PROT without the protein sequences used to train the program . All motifs were detected with a mean average sensitivity of 0.98 and a mean average specificity of 0.92 . AVAILABILITY: The program is freely available for use on the internet at The user can find additional information at this site.

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